METHOD FOR DETECTING HUMAN PAPILLOMAVIRUS mRNA

ABSTRACT

An in vitro method is provided for screening human female subjects to assess their risk of developing cervical carcinoma which comprises screening the subject for expression of mRNA transcripts from the E6 and optionally the L1 gene of human papillomavirus, wherein subjects positive for expression of L1 and/or E6 mRNA are scored as being at risk of developing cervical carcinoma. Kits for carrying out such methods are also provided.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/500,832, filed Jan. 6, 2005 and now pending, which is a nationalstage filing under 35 U.S.C. § 371 of PCT International applicationPCT/GB2003/000034, filed Jan. 7, 2003, which was published under PCTArticle 21(2) in English.

FIELD OF THE INVENTION

The present invention relates to in vitro methods of screening humansubjects in order to assess their risk of developing cervical carcinoma.

BACKGROUND TO THE INVENTION

Cervical carcinoma is one of the most common malignant diseasesworld-wide and is one of the leading causes of morbidity and mortalityamong women (Parkin D M, Pisani P, Ferlay J (1993) Int J Cancer 54:594-606; Pisani P, Parkin D M, Ferlay J (1993) Int J Cancer 55:891-903). 15,700 new cases of invasive cervical cancer were predicted inthe United States in 1996, and the annual world-wide incidence isestimated to be 450,000 by the World Health Organization (1990). Theannual incidence rate differs in different parts of the world, rangingfrom 7.6 per 100,000 in western Asia to 46.8 per 100,000 in southernAfrica (Parkin et al., 1993 ibid).

The current conception of cervical carcinoma is that it is a multistagedisease, often developing over a period of 10-25 years. Invasivesquamous-cell carcinoma of the cervix is represented by penetrationthrough the basal lamina and invading the stroma or epithelial laminapropria. The clinical course of cervical carcinoma shows considerablevariation. Prognosis has been related to clinical stage, lymph nodeinvolvement, primary tumour mass, histology type, depth of invasion andlymphatic permeation (Delgado G, et al., (1990) Gynecol Oncol 38:352-357). Some patients with less favourable tumour characteristics havea relatively good outcome, while others suffer a fatal outcome of aninitially limited disease. This shows a clear need for additionalmarkers to further characterise newly diagnosed cervical carcinomas, inorder to administer risk-adapted therapy (Ikenberg H, et al., Int. J.Cancer 59:322-6. 1994).

The epidemiology of cervical cancer has shown strong association withreligious, marital and sexual patterns. Almost 100 case-control studieshave examined the relationship between HPV and cervical neoplasia andalmost all have found positive associations (IARC monographs, 1995). Theassociation is strong, consistent and specific to a limited number ofviral types (Munoz N, Bosch F X (1992) HPV and cervical neoplasia:review of case-control and cohort studies. IARC Sci Publ 251-261). Amongthe most informative studies, strong associations with HPV 16 DNA havebeen observed with remarkable consistency for invasive cancer andhigh-grade CIN lesions, ruling out the possibility that this associationcan be explained by chance, bias or confounding (IARC monographs, 1995).Indirect evidence suggested that HPV DNA detected in cancer cells is agood marker for the role of HPV infection earlier in the carcinogenesis.Dose-response relationship has been reported between increasing viralload and risk of cervical carcinoma (Munoz and Bosch, 1992 ibid). Insome larger series up to 100% of the tumours were positive for HPV butthe existence of virus-negative cervical carcinomas is still debatable(Meijer C J, et al., (1992) Detection of human papillomavirus incervical scrapes by the polymerase chain reaction in relation tocytology: possible implications for cervical cancer screening. IARC SciPubl 271-281; Das B C, et al., (1993) Cancer 72: 147-153).

The most frequent HPV types found in squamous-cell cervical carcinomasare HPV 16 (41%-86%) and 18 (2%-22%). In addition HPV 31, 33, 35, 39,45, 51, 52, 54, 56, 58, 59, 61, 66 and 68 are also found (IARC,monographs, 1995). In the HPV2000 International conference in BarcelonaHPV 16, 18, 31 and 45 were defined as high risk, while HPV 33, 35, 39,51, 52, 56, 58, 59, 68 were defined as intermediate risk (Keerti V.Shah. P71). The 13 high risk plus intermediate risk HPVs are togetheroften referred to as cancer-associated HPV types.

A number of studies have explored the potential role of HPV testing incervical screening (see Cuzick et al. A systematic review of the role ofhuman papillomavirus testing within a cervical screening programme.Health Technol Assess 3:14. 1999).

Reid et al., (Reid R, et al., (1991) Am J Obstet Gynecol 164: 1461-1469)where the first to demonstrate a role for HPV testing in a screeningcontext. This study was carried out on high-risk women from sexuallytransmitted disease clinics and specialist gynaecologists, and used asensitive (low stringency) Southern blot hybridisation for HPVdetection. A total of 1012 women were enrolled, and cervicography wasalso considered as a possible adjunct to cytology. Twenty-three CINII/III lesions were found altogether, but only 12 were detected bycytology (sensitivity 52%, specificity 92%). HPV testing found 16high-grade lesions.

Bauer et al. (Bauer H M, et al., (1991) JAMA 265: 472-477) report anearly PCR-based study using MY09/11 primers (Manos M, et al., (1990)Lancet 335: 734) in young women attending for routine smears (collegestudents). They found a positive rate of 46% in 467 women, which wasmuch higher than for dot blot assay (11%).

In a study using PCR with GP5/6 primers (Van Den Brule A J, et al.,(1990) J Clin Microbiol 28: 2739-2743) van der Brule et al. (Van DenBrule A J, et al., (1991) Int J Cancer 48: 404-408) showed a very strongcorrelation of HPV positivity with cervical neoplasia as assessed bycytology. In older women (aged 35-55 years) with negative cytology theHPV positivity rate was only 3.5%, and this was reduced to 1.5% if onlytypes 16, 18, 31 and 33 were considered, while women with histologicalcarcinoma in situ were all HPV-positive, and 90% had one of the fourabove types. Women with less severe cytological abnormalities had lowerHPV positivity rates in a graded way, showing a clear trend.

Roda Housman et al. (Roda Housman A M, et al., (1994) Int J Cancer 56:802-806) expanded these observations by looking at a further 1373 womenwith abnormal smears. This study also confirmed increasing positivityrate with increasing severity of smear results. They also noted that thelevel of HPV heterogeneity decreased from 22 types for low-grade smearsto ten “high-risk” types for high grade smears. This paper did notinclude any cytologically negative women, nor was cytological diseaseconfirmed histologically.

Cuzick et al. (Cuzick J, et al., (1992) Lancet 340: 112-113; Cuzick J,et al., (1994) Br J Cancer 69: 167-171) were the first to report thatHPV testing provided useful information for the triage of cytologicalabnormalities detected during random screening. In a study of 133 women,referral for coloposcopy they found a positive predictive value of 42%,which was similar to that for moderate dyskaryosis. The results weremost striking for HPV 16, where 39 of 42 HPV 16 positive women werefound to have high-grade CIN on biopsy. This study pointed out theimportance of assessing viral load and only considered high levels ofhigh-risk types as positive.

Cox et al. (Cox J T, et al., (1995) Am J Obstet Gynecol 172: 946-954)demonstrated a role for HPV testing using the Hybrid Capture™ system(DIGENE Corporation, Gaithersburg, Md., USA) for triaging women withborderline smears. This test was performed on 217 such women from acollege referral service, and a sensitivity of 93% was found forCINII/III compared with 73% for repeat cytology. High viral load wasfound to further improve performance by reducing false positives. When 5RLU was taken as a cut-off, a PPV of approximately 24% was found with noloss of sensitivity.

Cuzick et al. (Cuzick J, et al., (1995) Lancet 345: 1533-1536) evaluatedHPV testing in a primary screening context in 1985 women attending forroutine screening at a family planning clinic. Sensitivity usingtype-specific PCR for the four common HPV types (75%) exceeded that ofcytology (46%), and the PPV for a positive HPV test (42%) was similar tothat for moderate dyskaryosis (43%).

WO 91/08312 describes methods for determining the prognosis ofindividuals infected with HPV which comprise measuring the level of HPVactivity by detecting transcripts of all or a portion of the E6 and/orE7 HPV genes in a sample and comparing the measurements of HPV activitywith a previously established relationship between activity and risk ofprogression to serious cervical dysplasia or carcinoma.

WO 99/29890 describes methods for the assessment of HPV infection basedon the measurement and analysis of gene expression levels. Inparticular, WO 99/29890 describes methods which are based on measuringthe levels of expression of two or more HPV genes (e.g. HPV E6, E7, L1and E2) and then comparing the ratio of expression of combinations ofthese genes to provide an indication of the stage of HPV-based diseasein a patient.

The present inventors have determined that it is possible to make aclinically useful assessment of HPV-associated disease based only on asimple positive/negative determination of expression of HPV L1 and E6mRNA transcripts, with no requirement for accurate quantitativemeasurements of expression levels or for determination of differences inthe levels of expression of the two transcripts. This method istechnically simple and, in a preferred embodiment, is amenable toautomation in a mid-to-high throughput format. Furthermore, on the basisof results obtained using the method of the invention the inventors havedefined a novel scheme for classification of patients on the basis ofrisk of developing cervical carcinoma which is related todisease-relevant molecular changes in the pattern of HPV gene expressionand is independent of CIN classification.

Therefore, in a first aspect the invention provides an in vitro methodof screening human subjects to assess their risk of developing cervicalcarcinoma which comprises screening for expression of mRNA transcriptsfrom the L1 gene and the E6 gene of human papillomavirus, whereinsubjects positive for expression of L1 and/or full length E6 mRNA arescored as being at risk of developing cervical carcinoma.

A positive screening result in the method of the invention is indicatedby positive expression of L1 mRNA and/or E6 mRNA in cells of the cervix.Positive expression of either one of these mRNAs or both mRNAs is takenas an indication that the subject is “at risk” for development ofcervical carcinoma. Women who express E6 mRNA are at high risk ofdeveloping cell changes because oncogenic E6 and E7 bind to cell cycleregulatory proteins and act as a switch for cell proliferation. Clearexpression of E6 mRNA provides a direct indication of cell changes inthe cervix. Expression of L1 mRNA, with or without expression of E6 mRNAis also indicative of the presence of an active HPV.

In the wider context of cervical screening, women identified as positivefor L1 and/or E6 mRNA expression may be selected for furtherinvestigation, for example using cytology. Thus, at one level the methodof the invention may provide a technical simple means of pre-screening apopulation of women in order to identify HPV-positive subjects who maybe selected for further investigation.

In a specific embodiment, the method of the invention may be used toclassify subjects into four different classes of risk for developingcervical carcinoma on the basis of positive/negative scoring ofexpression of L1 and E6 mRNA.

Accordingly, in a further aspect the invention provides an in vitromethod of screening human subjects to assess their risk of developingcervical carcinoma which comprises screening the subject for expressionof mRNA transcripts of the L1 gene of HPV and mRNA transcripts of the E6gene of HPV, and sorting the subject into one of four categories of riskfor development of cervical carcinoma based on expression of L1 and/orE6 mRNA according to the following classification:

Risk category 1: subjects negative for expression of L1 mRNA butpositive for expression of E6 mRNA from at least one of HPV types 16,18, 31, 33, 35, 39, 45, 52, 56, 58, 59, 66 or 68. Those individualspositive for expression of E6 mRNA from at least one of HPV types 16,18, 31 or 33 are scored as being at higher risk, for example incomparison to individuals negative for these types but positive forexpression of E6 mRNA from at least one of HPV types 35, 39, 45, 52, 56,58, 59, 66 or 68.Risk category 2: subjects positive for expression of L1 mRNA andpositive for expression of E6 mRNA from at least one of HPV types 16,18, 31, 33, 35, 39, 45, 52, 56, 58, 59, 66 or 68. Those individualspositive for expression of E6 mRNA from at least one of HPV types 16,18, 31 or 33 are scored as being at higher risk, for example incomparison to individuals negative for these types but positive forexpression of E6 mRNA from at least one of HPV types 35, 39, 45, 52, 56,58, 59, 66 or 68.Risk category 3: subjects positive for expression of L1 mRNA butnegative for expression of E6 mRNA from the cancer-associated HPV types,(e.g. negative for expression of E6 mRNA from HPV types 16, 18, 31, 33,35, 39, 45, 52, 56, 58, 59, 66 and 68).Risk category 4: subjects negative for expression of L1 mRNA andnegative for expression of E6 mRNA.

In a preferred embodiment, positive expression is indicated by thepresence of more than 50 copies of the transcript per ml (or totalvolume of the sample) and negative expression is indicated by thepresence of less than 1 copy of the transcript per ml (or total volumeof the sample).

The above classification is based on molecular events which are relevantto risk of developing cervical carcinoma and is independent of the CINstatus of the subjects. Thus, this method of classification may providean alternative to the use of cytology in the routine screening of womento identify those at potential risk of developing cervical carcinoma.The method may also be used as an adjunct to cytology, for example as aconfirmatory test to confirm a risk assessment made on the basis ofcytology.

Women positive for expression of high risk E6 mRNA from one of HPV types16, 18, 31 or 33 but negative for expression of L1 are in the highestlevel of risk of developing severe cell changes and cell abnormalities.This is due to the fact that a negative result for L1 mRNA expression isdirectly indicative of integrated HPV, and therefore a higherprobability of high and constant expression of E6 and E7. Integration ofa virus in the human genome has also a direct impact on the stability ofthe cells. Integration of HPV also reduces the possibility of regressionof cell changes.

Women positive for expression of E6 mRNA from one of HPV types 16, 18,31 or 33 and positive for expression of L1 mRNA have a “high risk” HPVexpression and it is still possible that the HPV has been integrated.However, the risk of these women is not classed as high as those who areL1 negative and E6 positive, since there is a reasonable probabilitythat they do not have integrated HPV.

Women negative for expression of E6 mRNA from HPV types 16, 18, 31 or 33but positive for expression of E6 mRNA from another HPV type, e.g. 35,39, 45, 52, 56, 58, 59, 66 and 68, are still considered “at risk” andmay therefore be placed in risk categories 1 or 2 (as defined above)depending on whether they are positive or negative for expression of L1mRNA.

Women positive for L1 mRNA but negative for E6 mRNA are scored as beingat moderate risk. There may be high-risk HPV types in the sample and L1expression is indicative of lytic activity. There may also be integratedHPV types but only with viruses that are rare. However, detection oflytic activity may show that the cell may soon develop some changes.

In the wider context of cervical screening the method of the inventionmay be used to classify women according to risk of developing cervicalcarcinoma and therefore provide a basis for decisions concerningtreatment and/or further screening. By way of example: women in riskcategory 1, particularly those who exhibit positive expression of E6mRNA from at least one of HPV types 16, 18, 31 or 33, might beidentified as requiring “immediate action”, meaning conisation orcolposcopy, including a biopsy and histology.

Women in risk category 2, as defined above, might be scored as requiringimmediate attention, meaning colposcopy alone or colposcopy including abiopsy and histology.

Women in risk category 3, as defined above, might be scored as requiringimmediate re-test, meaning recall for a further test for HPV expressionimmediately or after a relatively short interval, e.g. six months.

Women in risk category 4, as defined above, might be returned to thescreening program, to be re-tested for HPV expression at a later date.

In a further embodiment the invention provides an in vitro method ofscreening human subjects for the presence of integrated HPV or amodified episomal HPV genome, which method comprises screening thesubject for expression of mRNA transcripts from the L1 gene and the E6gene of human papillomavirus, wherein subjects negative for expressionof L1 mRNA but positive for expression of E6 mRNA are scored as carryingintegrated HPV.

The term “integrated HPV” refers to an HPV genome which is integratedinto the human genome.

The term “modified episomal HPV genome” is taken to mean an HPV genomewhich is retained within a cell of the human subject as an episome, i.e.not integrated into the human genome, and which carries a modificationas compared to the equivalent wild-type HPV genome, which modificationleads to constitutive or persistent expression of transcripts of the E6and/or E7 genes. The “modification” will typically be a deletion, amultimerisation or concatermerisation of the episome, a re-arrangementof the episome etc affecting the regulation of E6/E7 expression.

As aforesaid, the presence of integrated HPV or a modified episomal HPVgenome is indicated by a negative result for L1 mRNA expression,together with a positive result for expression of E6 mRNA in cells ofthe cervix. Therefore, the ability to predict the presence of integratedHPV or a modified episomal HPV genome in this assay is criticallydependent on the ability to score a negative result for L1 mRNAexpression. This requires a detection technique which has maximalsensitivity, yet produces minimal false-negative results. In a preferredembodiment this is achieved by using a sensitive amplification andreal-time detection technique to screen for the presence or absence ofL1 mRNA. The most preferred technique is real-time NASBA amplificationusing molecular beacons probes, as described by Leone et al., NucleicAcids Research., 1998, Vol 26, 2150-2155. Due to the sensitivity of thistechnique the occurrence of false-negative results is minimised and aresult of “negative L1 expression” can be scored with greaterconfidence.

In a further embodiment, a method of screening human subjects for thepresence of integrated HPV or a modified episomal HPV genome may bebased on screening for expression of E6 mRNA alone. Thus, the inventionrelates to an in vitro method of screening human subjects for thepresence of integrated HPV or a modified episomal HPV genome, whichmethod comprises screening the subject for expression of mRNAtranscripts from the E6 gene of human papillomavirus, wherein subjectspositive for expression of E6 mRNA are scored as carrying integrated HPVor a modified episomal HPV genome.

Moreover, individuals may be sorted into one of two categories of riskfor development of cervical carcinoma based on an “on/off” determinationof expression of E6 mRNA alone. Therefore, the invention provides an invitro method of screening human subjects to assess their risk ofdeveloping cervical carcinoma, which method comprises screening thesubject for expression of mRNA transcripts of the E6 gene of HPV andsorting the subject into one of two categories of risk for developmentof cervical carcinoma based on expression of E6 mRNA, whereinindividuals positive for expression of E6 mRNA are scored as carryingintegrated HPV or a modified episomal HPV genome and are thereforeclassified as “high risk” for development of cervical carcinoma, whereasindividuals negative for expression of E6 mRNA are scored as notcarrying integrated HPV or a modified episomal HPV genome and aretherefore classified as “no detectable risk” for development of cervicalcarcinoma.

Subjects are sorted into one of two categories of risk for developmentof cervical carcinoma based on an “on/off” determination of expressionof E6 mRNA in cells of the cervix. Individuals positive for expressionof E6 mRNA are scored as carrying integrated HPV or a modified episomalHPV genome and are therefore classified “high risk” for development ofcervical carcinoma, whereas individuals negative for expression of E6mRNA are scored as not carrying integrated HPV a modified episomal HPVgenome and are therefore classified as “no detectable risk” fordevelopment of cervical carcinoma.

In the context of cervical screening classification of subjects into thetwo groups having “high risk” or “no detectable risk” for development ofcervical carcinoma provides a basis for decisions concerning treatmentand/or further screening. For example subjects in the high risk categorymay be scored as requiring immediate further analysis, e.g. byhistological colposcopy, whilst those in the no detectable risk categorymay be referred back to the screening program at three or five yearintervals.

These methods are particularly useful for assessing risk of developingcarcinoma in subjects known to be infected with HPV, e.g. those testingpositive for HPV DNA, or subjects who have previously manifested acervical abnormality via cytology or pap smear. Subjects placed in the“no detectable risk” category on the basis of E6 mRNA expression mayhave HPV DNA present but the negative result for E6 expression indicatesthat HPV is unrelated to oncogene activity at the time of testing.

The presence of integrated HPV or a modified episomal HPV genome, asindicated by a positive result for E6 mRNA expression, is itselfindicative that the subject has abnormal cell changes in the cervix.Therefore, the invention also relates to an in vitro method ofidentifying human subjects having abnormal cell changes in the cervix,which method comprises screening the subject for expression of mRNAtranscripts of the E6 gene of HPV, wherein individuals positive forexpression of E6 mRNA are identified as having abnormal cell changes inthe cervix.

The term “abnormal cell changes in the cervix” encompasses cell changeswhich are characteristic of more severe disease than low-grade cervicallesions or low squamous intraepithelial lesions, includes cell changeswhich are characteristic of disease of equal or greater severity thanhigh-grade CIN (defined as a neoplastic expansion of transformed cells),CIN (cervical intraepithelial neoplasia) III, or high squamousintraepithelial neoplasia (HSIL), including lesions with multiploid DNAprofile and “malignant” CIN lesions with increased mean DNA-indexvalues, high percentage of DNA-aneuploidy and 2.5c Exceeding Rates(Hanselaar et al., 1992, Anal Cell Pathol.,

4:315-324; Rihet et al., 1996, J. Clin Pathol 49:892-896; and McDermottet al., 1997, Br. J. Obstet. Gynaecol. 104:623-625).

Cervical Intraepithelial Neoplasia (abbreviated “CIN”), also calledCervical Dysplasia, is a cervical condition caused Human PapillomaVirus. CIN is classified as I, II or III depending on its severity. Itis considered a pre-cancerous abnormality, but not an actual cancer. Themildest form, CIN I, usually goes away on its own, although rarely itcan progress to cancer. The more severe forms, CIN II and CIN III, mostoften stay the same or get worse with time. They can become a cancer,but almost never do if treated adequately.

HPV has been identified as a causative agent in development of cellularchanges in the cervix, which may lead to the development of cervicalcarcinoma. These cellular changes are associated with constitutive orpersistent expression of E6/E7 proteins from the HPV viral genome. Thus,it is possible to conclude that subjects in which expression of E6 mRNAcan be detected, particularly those subjects who exhibit persistent E6expression when assessed over a period of time, already manifestcellular changes in the cervix. These changes may have taken place inonly a very few cells of the cervix, and may not be detectable byconventional cytology. Nevertheless, with the use of sensitive, specificand accurate methods for detection of E6 mRNA it is possible to identifythose subjects who already exhibit cellular changes in the cervix at amuch earlier stage than would be possible using conventional cytologicalscreening. This will allow earlier intervention with treatments aimed atpreventing the development of cervical carcinoma.

As a result of HPV integration into the human genome or as a result ofthe “modification” in a modified episomal HPV genome, normal control ofthe viral E6/E7 oncogene transcription is lost (Durst et al., 1985, JGen Virol, 66(Pt 7): 1515-1522; Pater and Pater, 1985 Virology145:313-318; Schwarz et al., 1985, Nature 314: 111-114; Park et al.,1997, ibid). In contrast, in premalignant lesions and HPV-infectednormal epithelium papillomaviruses predominate in “unmodified” episomalforms, hence oncogene (E6/E7) transcription may be absent or efficientlydown-regulated (Johnson et al., 1990, J Gen Virol, 71(Pt 7): 1473-1479;Falcinelli et al., 1993, J Med Virol, 40: 261-265). Integration of humanpapillomavirus type 16 DNA into the human genome is observed to lead toa more unstable cell activity/genome, and increased stability of E6 andE7 mRNAs (Jeon and Lambert, 1995, Proc Natl Acad Sci USA 92: 1654-1658).Thus HPV integration, typically found in cervical cancers but onlyinfrequently found in CIN lesions (Carmody et al., 1996, Mol CellProbes, 10: 107-116), appears to be an important event in cervicalcarcinogenesis.

The present methods detect E6/E7 viral mRNA expression in the cervixinstead of DNA. E6/E7 viral expression in cervical cells is a much moreaccurate assessment of the risk of developing cancer than simply showingthat the HPV virus is present. Furthermore, the detection of HPVoncogene transcripts may be a more sensitive indicator of the directinvolvement of viral oncogenes in carcinogenesis (Rose et al., 1994,Gynecol Oncol, 52: 212-217; Rose et al., 1995, Gynecol Oncol, 56:239-244). Detection of E6/E7 transcripts by amplification and detectionis a useful diagnostic tool for risk evaluations regarding thedevelopment of CIN and its progression to cervical cancer, especially inhigh-risk HPV type-infected patients with ASCUS and CIN I (Sotlar etal., 1998, Gynecol Oncol, 69: 114-121; Selinka et al., 1998, Lab Invest,78: 9-18).

The expression of E6/E7 transcripts of HPV-16/18 is uniformly correlatedwith the physical status of HPV DNAs (Park et al., 1997, Gynecol Oncol,Vol: 65(1), 121-9). In most cervical carcinoma cells the E6 and E7 genesof specific human papillomaviruses are transcribed from viral sequencesintegrated into host cell chromosomes (von Kleben Doeberitz et al.,1991, Proc Natl Acad Sci USA. Vol: 88(4), 1411-5). Viral load andintegration has been evaluated in a large series of CIN lesions(Pietsaro et al., 2002, J Clin Microbiol, Vol: 40(3), 886-91). Only onesample contained exclusively episomal HPV16 DNA, and this lesionregressed spontaneously. Seventeen of 37 invasive cervical carcinomasamples were identified previously as containing the completelyintegrated HPV16 genome by using PCR covering the entire E1/E2 gene, andthis was confirmed by rliPCR in 16 cases. One case, however, showed alow level of episomal deoxyribonucleic acid in addition to thepredominant integrated form. Of the remaining 20 carcinoma samplesshowing episomal forms in the previous analysis, 14 were found tocontain integrated forms using rliPCR, and four contained multimeric(modified) episomal forms. Thus, in total, 31 of 37 of the carcinomas(84%) showed integrated HPV16 genome, while absence of integration couldnot be detected. (Kalantari et al, 2001, Diagn Mol Pathol, Vol: 10(1),46-54).

There have been virtually no observations that cervical carcinoma cellsexist without integrated HPV or modified episomal HPV DNA (Kalantari etal. 2001; Pietsaro et al., 2002, ibid). It has further been shown thatE6 and E7 may only be transcribed from integrated or modified episomalHPV DNA (von Kleben Doeberitz et al., 1991, ibid). Therefore, theinventors surmise that detection of E6/E7 expression provides a directindication of integrated HPV or modified episomal HPV and high oncogeneactivity, and conclude that in a clinical context detection of E6(E6/E7) expression alone is sufficient to identify subjects at “highrisk” of developing cervical carcinoma. In other words, if E6/E7 mRNAexpression can be detected in a cervical sample, this is directlyindicative of cellular abnormalities in the cervix and there is a veryhigh risk of development of cervical carcinoma due to persistent HPVoncogene activity. Therefore, detection of E6/E7 mRNA in a human subjectindicates that the subject has a very high risk of developing cervicalcarcinoma and should undergo immediate further screening, e.g. bycolposcopy.

If HPV E6/E7 mRNA expression is not detected, the subject may still havean HPV infection. However due to absence of integration and oncogeneactivity, it may regress spontaneously (as observed by Pietsaro et al.,2002, ibid).

In a clinical context the performance of methods which rely on screeningfor expression of E6 mRNA alone is critically dependent on the abilityto score a negative result for E6 mRNA expression with confidence. Thisagain requires a detection technique which has maximal sensitivity, yetproduces minimal false-negative results. In a preferred embodiment thisis achieved by using a sensitive amplification and real-time detectiontechnique to screen for the presence or absence of E6 mRNA. The mostpreferred technique is real-time NASBA amplification using molecularbeacons probes, as described by Leone et al., Nucleic Acids Research.,1998, Vol 26, 2150-2155. Due to the sensitivity of this technique theoccurrence of false-negative results is minimised and a result of“negative E6 expression” can be scored with greater confidence. This isextremely important if the assays are to be used in the context of aclinical screening program.

In the methods based on detection of E6 mRNA alone it is preferred todetect at least types HPV 16, 18, 31, 33 and 45, and in a preferredembodiment the assay may detect only these HPV types. DNA from HPV types16, 18, 31 and 33 has been detected in more than 87% of cervicalcarcinoma samples (Karlsen et al., 1996, J Clin Microbiol,34:2095-2100). Other studies have shown that E6 and E7 are almostinvariably retained in cervical cancers, as their expression is likelyto be necessary for conversion to and maintenance of the malignant state(Choo et al., 1987, J Med Virol 21:101-107; Durst et al., 1995, CancerGenet Cytogenet, 85: 105-112). In contrast to HPV detection systemswhich are based on detection of the undamaged genome or the L1 genesequence, detection of HPV mRNA expressed from the E6/E7 area may detectmore than 90% of the patients directly related to a risk of developingcervical carcinoma.

In the clinic, methods based on detection of E6 mRNA are preferred foruse in post-screening, i.e. further analysis of individuals having aprevious diagnosis of ASCUS, CIN 1 or Condyloma. The method may be usedto select those with a high risk of developing cervical carcinoma fromamongst the group of individuals having a previous diagnosis of ASCUS,CIN 1 or Condyloma. ASCUS, Condyloma and CIN I may be defined as more orless the same diagnosis due to very low reproducibility betweendifferent cytologists and different cytological departments. Östör (IntJ. Gyn Path. 12:186-192. 1993) found that only around 1% of the CIN 1cases may progress to cervical carcinoma. Thus, there is a genuine needfor an efficient method of identifying the subset of individuals withASCUS, Condyloma or CIN I who are at substantial risk of developingcervical carcinoma. One of HPV types 16, 18, 31 or 33 was detected in87% of the cervical carcinoma cases study by Karlsen et al., 1996. Byinclusion of HPV 45, nearly 90% of the cervical carcinoma samples arefound to be related to these five HPV types. Therefore, calculated fromthe data provided by Östör (Int J. Gyn Path. 12:186-192. 1993) more than99.9% are detected cases with ASCUS, CIN I or condyloma are missed byour HPV-Proofer kit.

In the methods of the invention “positive expression” of an mRNA istaken to mean expression above background. There is no absoluterequirement for accurate quantitative determination of the level of mRNAexpression or for accurate determination of the relative levels ofexpression of L1 and E6 mRNA.

In certain embodiments, the methods of the invention may comprise aquantitative determination of levels of mRNA expression. In a preferredembodiment in order to provide a clear distinction between “positiveexpression” and “negative expression” a determination of “positiveexpression” may require the presence of more than 50 copies of therelevant mRNA (per ml of sample or per total volume of sample), whereasa determination of “negative expression” may require the presence ofless than 1 copy of the relevant mRNA (per ml of sample or per totalvolume of sample).

The methods of the invention will preferably involve screening for E6mRNA using a technique which is able to detect specifically E6 mRNA fromcancer-associated HPV types, more preferably “high risk”cancer-associated HPV types. In the most preferred embodiment themethods involve screening for E6 mRNA using a technique which is able todetect E6 mRNA from HPV types 16, 18, 31 and 33, and preferably also 45.Most preferably, the method will specifically detect expression of E6mRNA from at least one of HPV types 16, 18, 31, 33, and preferably also45, and most preferably all five types. However, women positive forpositive for expression of E6 from other types than 16, 18, 31, 33 and45, e.g. 35, 39, 45, 52, 56, 58, 59, 66 and 68 may still be “at risk” ofdeveloping cervical carcinoma. Thus, the method may encompass screeningfor expression of E6 mRNA from one or more of these HPV types, mostpreferably in addition to screening for E6 mRNA from HPV types 16, 18,31, 33 and 45. Certain HPV types exhibit a markedgeographical/population distribution. Therefore, it may be appropriateto include primers specific for an HPV type known to be prevalent in thepopulation/geographical area under test, for example in addition toscreening for HPV types 16, 18, 31, 33 and 45.

For the avoidance of doubt, unless otherwise stated the term “E6 mRNA”as used herein encompasses all naturally occurring mRNA transcriptswhich contain all or part of the E6 open reading frame, includingnaturally occurring splice variants, and therefore includes transcriptswhich additionally contain all or part of the E7 open reading frame (andindeed further open reading frames). The terms “E6/E7 mRNA”, “E6/E7transcripts” etc are used interchangeably with the terms “E6 mRNA”, “E6transcripts” and also encompass naturally occurring mRNA transcriptswhich contain all or part of the E6 open reading frame, includingnaturally occurring splice variants, and transcripts which contain allor part of the E7 open reading frame. The term “oncogene expression”,unless otherwise stated, also refers to naturally occurring mRNAtranscripts which contain all or part of the E6 open reading frame,including naturally occurring splice variants, and transcripts whichcontain all or part of the E7 open reading frame.

Four E6/E7 mRNA species have so far been described in cells infectedwith HPV 16, namely an unspliced E6 transcript and three splicedtranscripts denoted E6*I, E6*II and E6*III (Smotkin D, et al., J. Virol.1989 March 63(3):1441-7; Smotkin D, Wettstein F O. Proc Natl Acad SciUSA. 1986 July 83(13):4680-4; Doorbar J. et al., Virology. 1990September 178(1):254-62; Cornelissen M T, et al. J Gen Virol. 1990 May71(Pt 5):1243-6; Johnson M A, et al. J Gen Virol. 1990 July 71(Pt7):1473-9; Schneider-Maunoury S, et al. J. Virol. 1987 October61(10):3295-8; Sherman L, et al. Int J. Cancer. 1992 February50(3):356-64). All four transcripts are transcribed from a singlepromoter (p97) located just upstream of the second ATG of the E6 ORF.

In one embodiment the methods may comprise screening for E6 transcriptswhich contain all or part of the E7 open reading frame, This may beaccomplished, for example, using primers or probes specific for the E7coding region.

In a further embodiment, the methods may comprise screening for thepresence of “full length” E6 transcripts. In the case of HPV 16 the term“full length E6 transcripts” refers to transcripts which contain all ofthe region from nucleotide (nt) 97 to nt 880 in the E6 ORF, inclusive ofnt 97 and 880. Nucleotide positions are numbered according to standardHPV nomenclature (see Human Papillomavirus Compendium OnLine, availablevia the internet or in paper form from HV Database, Mail Stop K710, LosAlamos National Laboratory, Los Alamos, N. Mex. 87545, USA). Specificdetection of full length transcripts may be accomplished, for example,using primers or probes which are specific for the region which ispresent only in full length E6 transcripts, not in splice variants.Different HPV types exhibit different patterns of E6/E7 mRNA expression.Transcript maps for various HPV types, including HPV types 16 and 31,which may be used to assist in the design of probes or primers fordetection of E6/E7 transcripts are publicly available via the HumanPapillomavirus Compendium (as above).

E6 oligonucleotide primers are described herein which are suitable foruse in amplification of regions of the E6 mRNA from various HPV types byNASBA or PCR.

In a preferred embodiment methods which involve screening for L1 mRNAexpression may comprise screening for L1 mRNA expression using atechnique which is able to detect L1 mRNA from substantially all knownHPV types or at least the major cancer-associated HPV types (e.g.preferably all of HPV types 16, 18, 31 and 33). L1 primers and probesare described herein which are capable of detecting L1 mRNA from HPVtypes 6, 11, 16, 18, 31, 33, 35 and 51 in cervical samples.

Detection of L1 transcripts can be said to detect HPV “virulence”,meaning the presence of HPV lytic activity. Detection of E6/E7transcripts can be said to detect HPV “pathogenesis” since expression ofthese mRNAs is indicative of molecular events associated with risk ofdeveloping carcinoma.

In a study of 4589 women it was possible to detect all except one caseof CIN III lesions or cancer using a method based on screening forexpression of E6 and L1 mRNA (see accompanying Examples).

In further embodiments, the above-described methods of the invention maycomprise screening for expression of mRNA transcripts from the humanp16^(ink4a) gene, in addition to screening for expression of HPV L1and/or E6 transcripts.

A positive result for expression of p16^(ink4a) mRNA is taken as afurther indication of risk of developing cervical carcinoma.

P16^(ink4a), and the related family members, may function to regulatethe phosphorylation and the growth suppressive activity of therestinoblastoma gene product (RB). In support of this, it has been foundthat there is an inverse relationship between the expression ofp16^(ink4a) protein and the presence of normal RB in selected cancercell lines; p16^(ink4a) protein is detectable when RB is mutant,deleted, or inactivated, and it is markedly reduced or absent in celllines that contain a normal RB. Kheif et al. (Kheif S N et al., Proc.Natl. Acad. Sci. USA 93:4350-4354. 1996), found that p16^(ink4a) proteinis expressed in human cervical carcinoma cells that contain either amutant RB or a wild-type RB that is functionally inactivated by E7. Theyalso show that the inactivation of RB correlates with an upregulation ofp16^(ink4a) confirming a feedback loop involving p16^(ink4a) and RB.Milde-Langosch et al. (Milde-Langosch K, et al., (2001) Virchows Arch439: 55-61) found that there were significant correlations betweenstrong p16 expression and HPV16/18 infection and between strong p16expression and HPV 16/18 E6/E7 oncogene expression. Klaes et al., (KlaesR, et al., (2001) Int J Cancer 92: 276-284) observed a strong overexpression of the p16^(ink4a) gene product in 150 of 152 high-gradedysplastic cervical lesions (CIN II to invasive cancer), whereas normalcervical epithelium or inflammatory or metaplastic lesions were notstained with the p16^(ink4a) specific monoclonal antibody E6H4. All CINI scored lesions associated with LR-HPV types displayed no or only focalor sporadic reactivity, whereas all but two CIN I scored lesionsassociated with HR-HPV types showed strong and diffuse staining forp16^(ink4a).

The disclosed screening methods may be carried out on a preparation ofnucleic acid isolated from a clinical sample or biopsy containingcervical cells taken from the subject under test. Suitable samples whichmay be used as a source of nucleic acid include (but not exclusively)cervical swabs, cervical biopsies, cervical scrapings, skinbiopsies/warts, also paraffin embedded tissues, and formalin or methanolfixed cells.

The preparation of nucleic acid to be screened using the disclosedmethod must include mRNA, however it need not be a preparation ofpurified poly A+ mRNA and preparations of total RNA or crudepreparations of total nucleic acid containing both RNA and genomic DNA,or even crude cell lysates are also suitable as starting material for aNASBA reaction. Essentially any technique known in the art for theisolation of a preparation of nucleic acid including mRNA may be used toisolate nucleic acid from a test sample. A preferred technique is the“Boom” isolation method described in U.S. Pat. No. 5,234,809 andEP-B-0389,063. This method, which can be used to isolate a nucleic acidpreparation containing both RNA and DNA, is based on the nucleic acidbinding properties of silicon dioxide particles in the presence of thechaotropic agent guanidine thiocyanate (GuSCN).

The methods of the invention are based on assessment of activetranscription of the HPV genome in cervical cells. The methods are notlimited with respect to the precise technique used to detect mRNAexpression. Many techniques for detection of specific mRNA sequences areknown in the art and may be used in accordance with the invention. Forexample, specific mRNAs may be detected by hybridisation, amplificationor sequencing techniques.

It is most preferred to detect mRNA expression by means of anamplification technique, most preferably an isothermal amplificationsuch as NASBA, transcription-mediated amplification, signal-mediatedamplification of RNA technology, isothermal solution phaseamplification, etc. All of these methods are well known in the art Morepreferably mRNA expression is detected by an isothermal amplification incombination with real-time detection of the amplification product. Themost preferred combination is amplification by NASBA, coupled withreal-time detection of the amplification product using molecular beaconstechnology, as described by Leone et al., Nucleic Acids Research, 1998,Vol 26, 2150-2155.

Methods for the detection of HPV in a test sample using the NASBAtechnique will generally comprise the following steps:

(a) assembling a reaction medium comprising suitable primer-pairs, anRNA directed DNA polymerase, a ribonuclease that hydrolyses the RNAstrand of an RNA-DNA hybrid without hydrolysing single or doublestranded RNA or DNA, an RNA polymerase that recognises said promoter,and ribonucleoside and deoxyribonucleoside triphosphates;

(b) incubating the reaction medium with a preparation of nucleic acidisolated from a test sample suspected of containing HPV under reactionconditions which permit a NASBA amplification reaction; and

(c) detecting and/or quantitatively measuring any HPV-specific productof the NASBA amplification reaction.

Detection of the specific product(s) of the NASBA reaction (i.e. senseand/or antisense copies of the target RNA) may be carried out in anumber of different ways. In one approach the NASBA product(s) may bedetected with the use of an HPV-specific hybridisation probe capable ofspecifically annealing to the NASBA product. The hybridisation probe maybe attached to a revealing label, for example a fluorescent,luminescent, radioactive or chemiluminescent compound or an enzyme labelor any other type of label known to those of ordinary skill in the art.The precise nature of the label is not critical, but it should becapable of producing a signal detectable by external means, either byitself or in conjunction with one or more additional substances (e.g.the substrate for an enzyme).

A preferred detection method is so-called “real-time NASBA” which allowscontinuous monitoring of the formation of the product of the NASBAreaction over the course of the reaction. In a preferred embodiment thismay be achieved using a “molecular beacons” probe comprising anHPV-specific sequence capable of annealing to the NASBA product, astem-duplex forming oligonucleotide sequence and a pair offluorescer/quencher moieties, as known in the art and described herein.If the molecular beacons probe is added to the reaction mixture prior toamplification it may be possible to monitor the formation of the NASBAproduct in real-time (Leone et al., Nucleic Acids Research, 1998, Vol26, 2150-2155). Reagent kits and instrumentation for performingreal-time NASBA detection are available commercially (e.g. NucliSens™EasyQ system, from Organon Teknika).

In a further approach, the molecular beacons technology may beincorporated into the primer 2 oligonucleotide allowing real-timemonitoring of the NASBA reaction without the need for a separatehybridisation probe.

In a still further approach the products of the NASBA reaction may bemonitored using a generic labelled detection probe which hybridises to anucleotide sequence in the 5′ terminus of the primer 2 oligonucleotide.This is equivalent to the “NucliSens™” detection system supplied byOrganon Teknika. In this system specificity for NASBA products derivedfrom the target HPV mRNA may be conferred by using HPV-specific captureprobes comprising probe oligonucleotides as described herein attached toa solid support such as a magnetic microbead. Most preferably thegeneric labelled detection probe is the ECL™ detection probe supplied byOrganon Teknika. NASBA amplicons are hybridized to the HPV-specificcapture probes and the generic ECL probe (via a complementary sequenceon primer 2). Following hybridization the bead/amplicon/ECL probecomplexes may be captured at the magnet electrode of an automatic ECLreader (e.g. the NucliSens™ reader supplied by Organon Teknika).Subsequently, a voltage pulse triggers the ECL™ reaction.

The detection of HPV mRNA is also of clinical relevance in cancers otherthan cervical carcinoma including, for example, head and neck carcinoma,oral and tongue carcinoma, skin carcinoma, anal and vaginal carcinoma.Detection of HPV mRNA may also be very useful in the diagnosis ofmicrometastases in lymph nodes in the lower part of the body. Hence, theinvention also contemplates screens for susceptibility to theabove-listed cancers based on screening for expression of HPV L1 and E6transcripts.

In accordance with a further aspect of the invention there is provided akit for use in the detection of transcripts of the L1 and E6 genes ofHPV, the kit comprising at least one primer-pair suitable for use inamplification of a region of L1 transcripts from at least HPV types 16,18, 31 and 33, and preferably also HPV 45, and one or more primer-pairswhich enable amplification of a region of E6 transcripts from HPV types16, 18, 31 and 33, and preferably also HPV 45.

“Primer-pair” taken to mean are pair of primers which may be used incombination to amplify a specific region of the L1 or E6 mRNA using anyknown nucleic acid technique. In preferred embodiments the primer-pairsincluded in the kit will be suitable for use in NASBA amplification orsimilar isothermal amplification techniques.

The individual primers making up each primer-pair included in the kitmay be supplied separately (e.g. a separate container of each primer)or, more preferably, may be supplied mixed in a single container.Combinations of two or more primer-pairs may be supplied ready-mixed ina single container within the kit. It may be convenient to supply two ormore primer-pairs in a single container where the two or moreamplification reactions are to be “multiplexed”, meaning performedsimultaneously in a single reaction vessel.

The primer-pair(s) suitable for use in amplification of a region of E6transcripts should enable amplification a region of E6 mRNA from atleast the major cancer-associated HPV types 16, 18, 31 and 33, andpreferably also HPV 45. There are several different ways in which thiscan be achieved.

In one embodiment, the kit may contain separate primer-pairs specificfor each of HPV types 16, 18, 31 and 33, and preferably also HPV 45.These primer-pairs may be supplied within the kit in separatecontainers, or they may be supplied as mixtures of two or moreprimer-pairs in a single container, for example to enable multiplexingof the amplification reactions.

In a further embodiment, the kit may contain a single primer-paircapable of amplifying a region of the E6 gene from HPV types 16, 18, 31and 33, and preferably also HPV 45, which thus enables amplification ofall four (preferably five) types in a single amplification reaction.This could, for example, be achieved with the use of a pair ofdegenerate primers or by selection of a region of the E6 mRNA which ishighly conserved across HPV types.

The E6 primer-pair may correspond to any region of the E6 mRNA, an mayenable amplification of all or part of the E6 open reading frame and/orthe E7 open reading frame.

The kit may further include primer-pairs suitable for use inamplification of E6 mRNA from HPV types other than types 16, 18, 31 and33, and preferably also HPV 45. For example, the kit may be supplementedwith E6 primers for detection of an HPV type which is endemic in aparticular geographical area or population.

The primer-pair(s) suitable for use in amplification of a region of L1transcripts should be capable of amplifying a region of L1 mRNA from atleast the major cancer-associated HPV types 16, 18, 31 and 33, andpreferably also HPV 45, and will preferably be suitable for use inamplification of a region of L1 mRNAs from substantially all known HPVtypes. With the use of such primers it is possible to test for activetranscription of L1 mRNA from multiple HPV types in a singleamplification reaction.

It is possible to design primers capable of detecting L1 transcriptsfrom multiple HPV types by selecting regions of the L1 transcript whichare highly conserved.

In a further approach, specificity for multiple HPV types may beachieved with the use of degenerate oligonucleotide primers or complexmixtures of polynucleotides which exhibit minor sequence variations,preferably corresponding to sites of sequence variation between HPVgenotypes. The rationale behind the use of such degenerate primers ormixtures is that the mixture may contain at least one primer-paircapable of detecting each HPV type.

In a still further approach specificity for multiple HPV types may beachieved by incorporating into the primers one or more inosinenucleotides, preferably at sites of sequence variation between HPVgenotypes.

The E6 and L1 primer-pairs may be supplied in separate containers withinthe kit, or the L1 primer-pair(s) may be supplied as a mixture with oneor more E6 primer-pairs in a single container.

The kits may further comprise one or more probes suitable for use indetection of the products of amplification reactions carried out usingthe primer-pairs included within the kit. The probe(s) may be suppliedas a separate reagent within the kit. Alternatively, the probe(s) may besupplied as a mixture with one or more primer-pairs.

The primers and probes included in the kit are preferably singlestranded DNA molecules. Non-natural synthetic polynucleotides whichretain the ability to base-pair with a complementary nucleic acidmolecule may also be used, including synthetic oligonucleotides whichincorporate modified bases and synthetic oligonucleotides wherein thelinks between individual nucleosides include bonds other thanphosphodiester bonds. The primers and probes may be produced accordingto techniques well known in the art, such as by chemical synthesis usingstandard apparatus and protocols for oligonucleotide synthesis.

The primers and probes will typically be isolated single-strandedpolynucleotides of no more than 100 bases in length, more typically lessthan 55 bases in length. For the avoidance of doubt it is hereby statedthat the terms “primer” and “probe” exclude naturally occurringfull-length HPV genomes.

Several general types of oligonucleotide primers and probesincorporating HPV-specific sequences may be included in the kit.Typically, such primers and probes may comprise additional, non-HPVsequences, for example sequences which are required for an amplificationreaction or which facilitate detection of the products of theamplification reaction.

The first type of primers are primer 1 oligonucleotides (also referredto herein as NASBA P1 primers), which are oligonucleotides of generallyapproximately 50 bases in length, containing an average of about 20bases at the 3′ end that are complementary to a region of the targetmRNA. Oligonucleotides suitable for use as NASBA P1 primers are denoted“P1/PCR” in Table 1. P1 primer oligonucleotides have the generalstructure X₁-SEQ, wherein SEQ represents an HPV-specific sequence and X₁is a sequence comprising a promoter that is recognized by a specific RNApolymerase. Bacteriophage promoters, for example the T7, T3 and SP6promoters, are preferred for use in the oligonucleotides of theinvention, since they provide advantages of high level transcriptionwhich is dependent only on binding of the appropriate RNA polymerase. Ina preferred embodiment, sequence “X₁” may comprise the sequenceAATTCTAATACGACTCACTATAGGG (SEQ ID No 171) or the sequenceAATTCTAATACGACTCACTATAGGGAGAAGG (SEQ ID No 172). These sequencescontains a T7 promoter, including the transcription initiation site forT7 RNA polymerase.

The HPV-specific sequences in the primers denoted in Table 1 as “P1/PCR”may also be adapted for use in standard PCR primers. When thesesequences are used as the basis of NASBA P1 primers they have thegeneral structure X₁-SEQ, as defined above. The promoter sequence X₁ isessential in a NASBA P1 primer. However, when the same sequences areused as the basis of standard PCR primers it is not necessary to includeX₁.

A second type of primers are NASBA primer 2 oligonucleotides (alsoreferred to herein as NASBA P2 primers) which generally comprise asequence of approximately 20 bases substantially identical to a regionof the target mRNA. The oligonucleotide sequences denoted in Table 1 as“P2/PCR” are suitable for use in both NASBA P2 primers and standard PCRprimers.

Oligonucleotides intended for use as NASBA P2 primers may, in aparticular but non-limiting embodiment, further comprise a sequence ofnucleotides at the 5′ end which is unrelated to the target mRNA butwhich is capable of hybridising to a generic detection probe. Thedetection probe will preferably be labelled, for example with afluorescent, luminescent or enzymatic label. In one embodiment thedetection probe is labelled with a label that permits detection usingECL™ technology, although it will be appreciated that the invention isin no way limited to this particular method of detection. In a preferredembodiment the 5′ end of the primer 2 oligonucleotides may comprise thesequence GATGCAAGGTCGCATATGAG (SEQ ID No 170). This sequence is capableof hybridising to a generic ECL™ probe commercially available fromOrganon Teknika having the following structure:

Ru(bpy)₃ ²⁺-GAT GCA AGG TCG CAT ATG AG-3′

In a different embodiment the primer 2 oligonucleotide may incorporate“molecular beacons” technology, which is known in the art and described,for example, in WO 95/13399 by Tyagi and Kramer, Nature Biotechnology.14: 303-308, 1996, to allow for real-time monitoring of the NASBAreaction.

Target-specific probe oligonucleotides may also be included within thekit. Probe oligonucleotides generally comprise a sequence ofapproximately 20-25 bases substantially identical to a region of thetarget mRNA, or the complement thereof. Example HPV-specificoligonucleotide sequences which are suitable for use as probes aredenoted “PO” in Table 1. The probe oligonucleotides may be used astarget-specific hybridisation probes for detection of the products of aNASBA or PCR reaction. In this connection the probe oligonucleotides maybe coupled to a solid support, such as paramagnetic beads, to form acapture probe (see below). In a preferred embodiment the 5′ end of theprobe oligonucleotide may be labelled with biotin. The addition of abiotin label facilitates attachment of the probe to a solid support viaa biotin/streptavidin or biotin/avidin linkage.

Target-specific probes enabling real-time detection of amplificationproducts may incorporate “molecular beacons” technology which is knownin the art and described, for example, by Tyagi and Kramer, NatureBiotechnology. 14: 303-308, 1996 and in WO 95/13399. ExampleHPV-specific oligonucleotide sequences suitable for use as molecularbeacons probes are denoted “MB” in Table 1.

The term “molecular beacons probes” as used herein is taken to meanmolecules having the structure:

X₂-arm₁-target-arm₂-X₃

wherein “target” represents a target-specific sequence of nucleotides,“X₂” and “X₃” represent a fluorescent moiety and a quencher moietycapable of substantially or completely quenching the fluorescence fromthe fluorescent moiety when the two are held together in close proximityand “arm₁” and “arm₂” represent complementary sequences capable offorming a stem duplex.

Preferred combinations of “arm₁” and “arm₂” sequences are as follows,however these are intended to be illustrative rather than limiting tothe invention:

cgcatg-SEQ-catgcg ccagct-SEQ-agctgg cacgc-SEQ-gcgtg cgatcg-SEQ-cgatcgccgtcg-SEQ-cgacgg cggacc-SEQ-ggtccg ccgaagg-SEQ-ccttcggcacgtcg-SEQ-cgacgtg cgcagc-SEQ-gctgcg ccaagc-SEQ-gcttggccaagcg-SEQ-cgcttgg cccagc-SEQ-gctggg ccaaagc-SEQ-gctttggcctgc-SEQ-gcagg ccaccc-SEQ-gggtgg ccaagcc-SEQ-ggcttgg ccagcg-SEQ-cgctggcgcatg-SEQ-catgcg

The use of molecular beacons technology allows for real-time monitoringof amplification reactions, for example NASBA amplification (see Leoneet al., Nucleic Acids Research., 1998, vol: 26, pp 2150-2155). Themolecular beacons probes generally include complementary sequencesflanking the HPV-specific sequence, represented herein by the notationarm₁ and arm₂, which are capable of hybridising to each other form astem duplex structure. The precise sequences of arm₁ and arm₂ are notmaterial to the invention, except for the requirement that thesesequences must be capable of forming a stem duplex when the probe is notbound to a target HPV sequence.

Molecular beacons probes also include a fluorescent moiety and aquencher moiety, the fluorescent and the quencher moieties beingrepresented herein by the notation X₂ and X₃. As will be appreciated bethe skilled reader, the fluorescer and quencher moieties are selectedsuch that the quencher moiety is capable of substantially or completelyquenching the fluorescence from the fluorescent moiety when the twomoieties are in close proximity, e.g. when the probe is in the hairpin“closed” conformation in the absence of the target sequence. Uponbinding to the target sequence, the fluorescent and quencher moietiesare held apart such that the fluorescence of the fluorescent moiety isno longer quenched.

Many examples of suitable pairs of quencher/fluorescer moieties whichmay be used in accordance with the invention are known in the art (seeWO 95/13399, Tyagi and Kramer, ibid). A broad range of fluorophores inmany different colours made be used, including for example5-(2′-aminoethyl)aminonaphthalene-1-sulphonic acid (EDANS), fluorescein,FAM and Texas Red (see Tyagi, Bratu and Kramer, 1998, NatureBiotechnology, 16, 49-53. The use of probes labelled with differentcoloured fluorophores enables “multiplex” detection of two or moredifferent probes in a single reaction vessel. A preferred quencher is4-(4′-dimethylaminophenylazo)benzoic acid (DABCYL), a non-fluorescentchromophore, which serves as a “universal” quencher for a wide range offluorophores. The fluorescer and quencher moieties may be covalentlyattached to the probe in either orientation, either with the fluorescerat or near the 5′ end and the quencher at or near the 3′ end or viceversa. Protocols for the synthesis of molecular beacon probes are knownin the art. A detailed protocol for synthesis is provided in a paperentitled “Molecular Beacons: Hybridization Probes for Detection ofNucleic Acids in Homogenous Solutions” by Sanjay Tyagi et al.,Department of Molecular Genetics, Public Health Research Institute, 455First Avenue, New York, N.Y. 10016, USA, which is available online viathe PHRI website (at www.phri.nyu.edu or www.molecular-beacons.org)

Suitable combinations of the NASBA P1 and NASBA P2 primers may be usedto drive a NASBA amplification reaction. In order to drive a NASBAamplification reaction the primer 1 and primer 2 oligonucleotides mustbe capable of priming synthesis of a double-stranded DNA from a targetregion of mRNA. For this to occur the primer 1 and primer 2oligonucleotides must comprise target-specific sequences which arecomplementary to regions of the sense and the antisense strand of thetarget mRNA, respectively.

In the first phase of the NASBA amplification cycle, the so-called“non-cyclic” phase, the primer 1 oligonucleotide anneals to acomplementary sequence in the target mRNA and its 3′ end is extended bythe action of an RNA-dependent DNA polymerase (e.g. reversetranscriptase) to form a first-strand cDNA synthesis. The RNA strand ofthe resulting RNA:DNA hybrid is then digested, e.g. by the action ofRNaseH, to leave a single stranded DNA. The primer 2 oligonucleotideanneals to a complementary sequence towards the 3′ end of this singlestranded DNA and its 3′ end is extended (by the action of reversetranscriptase), forming a double stranded DNA. RNA polymerase is thenable to transcribe multiple RNA copies from the now transcriptionallyactive promoter sequence within the double-stranded DNA. This RNAtranscript, which is antisense to the original target mRNA, can act as atemplate for a further round of NASBA reactions, with primer 2 annealingto the RNA and priming synthesis of the first cDNA strand and primer 1priming synthesis of the second cDNA strand. The general principles ofthe NASBA reaction are well known in the art (see Compton, J. Nature.350: 91-92).

The target-specific probe oligonucleotides described herein may also beattached to a solid support, such as magnetic microbeads, and used as“capture probes” to immobilise the product of the NASBA amplificationreaction (a single stranded RNA). The target-specific “molecularbeacons” probes described herein may be used for real-time monitoring ofthe NASBA reaction.

Kits according to the invention may also including a positive controlcontaining E6 and/or L1 mRNA from a known HPV type. Suitable controlsinclude, for example, nucleic acid extracts prepared from cell linesinfected with known HPV types (e.g. HeLa, CaSki).

Kits further may contain internal control amplification primers, e.g.primers specific for human U1A RNA.

Kits containing primers (and optionally probes) suitable for use inNASBA amplification may further comprise a mixture of enzymes requiredfor the NASBA reaction, e.g. enzyme mixture containing an RNA directedDNA polymerase (e.g. a reverse transcriptase), a ribonuclease thathydrolyses the RNA strand of an RNA-DNA hybrid without hydrolysingsingle or double stranded RNA or DNA (e.g. RNaseH) and an RNApolymerase. The RNA polymerase should be one which recognises thepromoter sequence present in the 5′ terminal region of the NASBA P1primers supplied in the reagent kit. The kit may also comprise a supplyof NASBA buffer containing the ribonucleosides and deoxyribonucleosidesrequired for RNA and DNA synthesis. The composition of a standard NASBAreaction buffer will be well known to those skilled in the art (see alsoLeone et al., ibid).

TABLE 1 E6-specific sequences for inclusion in NASBA/PCR primers andprobes Primer/ probe HPV SEQ ID type Sequence Type nt 1 P2/PCRCCACAGGAGCGACCCAGAAAGTTA 16 116 2 P1/PCR X₁-ACGGTTTGTTGTATTGCTGTTC 16368 3 P2/PCR CCACAGGAGCGACCCAGAAA 16 116 4 P1/PCRX₁-GGTTTGTTGTATTGCTGTTC 16 368 5 P1/PCR X₁-ATTCCCATCTCTATATACTA 16 258 6P1/PCR X₁-TCACGTCGCAGTAACTGT 16 208 7 P1/PCR X₁-TTGCTTGCAGTACACACA 16191 8 P1/PCR X₁-TGCAGTACACACATTCTA 16 186 9 P1/PCR X₁-GCAGTACACACATTCTAA16 185 10 P2/PCR ACAGTTATGCACAGAGCT 16 142 11 P2/PCR ATATTAGAATGTGTGTAC16 182 12 P2/PCR TTAGAATGTGTGTACTGC 16 185 13 P2/PCR GAATGTGTGTACTGCAAG16 188 14 PO ACAGTTATGCACAGAGCT 16 142 15 PO ATATTAGAATGTGTGTAC 16 18216 PO TTAGAATGTGTGTACTGC 16 185 17 PO GAATGTGTGTACTGCAAG 16 188 18 POCTTTGCTTTTCGGGATTTATGC 16 235 19 PO TATGACTTTGCTTTTCGGGA 16 230 20 MBX₂-arm₁-TATGACTTTGCTTTTCGGGA-arm₂-X₃ 16 230 21 P2/PCRCAGAGGAGGAGGATGAAATAGTA 16 656 22 P1/PCR X₁-GCACAACCGAAGCGTAGAGTCACAC 16741 23 PO TGGACAAGCAGAACCGGACAGAGC 16 687 24 P2/PCRCAGAGGAGGAGGATGAAATAGA 16 656 25 P1/PCR X₁-GCACAACCGAAGCGTAGAGTCA 16 74126 PO AGCAGAACCGGACAGAGCCCATTA 16 693 27 P2/PCR ACGATGAAATAGATGGAGTT 18702 28 P1/PCR X₁-CACGGACACACAAAGGACAG 18 869 28 PO AGCCGAACCACAACGTCACA18 748 30 P2/PCR GAAAACGATGAAATAGATGGAG 18 698 31 P1/PCRX₁-ACACCACGGACACACAAAGGACAG 18 869 32 PO GAACCACAACGTCACACAATG 18 752 33MB X₂-arm₁-GAACCACAACGTCACACAATG- 18 752 arm₂-X₃ 34 P2/PCRTTCCGGTTGACCTTCTATGT 18 651 35 P1/PCR X₁-GGTCGTCTGCTGAGCTTTCT 18 817 36P2/PCR GCAAGACATAGAAATAACCTG 18 179 37 P1/PCR X₁-ACCCAGTGTTAGTTAGTT 18379 38 PO TGCAAGACAGTATTGGAACT 18 207 39 P2/PCR GGAAATACCCTACGATGAAC 31164 40 P1/PCR X₁-GGACACAACGGTCTTTGACA 31 423 41 POATAGGGACGACACACCACACGGAG 31 268 42 P2/PCR GGAAATACCCTACGATGAACTA 31 16443 P1/PCR X₁-CTGGACACAACGGTCTTTGACA 31 423 44 PO TAGGGACGACACACCACACGGA31 269 45 P2/PCR ACTGACCTCCACTGTTATGA 31 617 46 P1/PCRX₁-TATCTACTTGTGTGCTCTGT 31 766 47 PO GACAAGCAGAACCGGACACATC 31 687 48P2/PCR TGACCTCCACTGTTATGAGCAATT 31 619 49 P1/PCRX₁-TGCGAATATCTACTTGTGTGCTCT GT 31 766 50 PO GGACAAGCAGAACCGGACACATCCAA31 686 51 MB X₂-arm₁-GGACAAGCAGAACCGGACACATCCAA- 31 686 arm₂-X₃ 52P2/PCR ACTGACCTCCACTGTTAT 31 617 53 P1/PCR X₁-CACGATTCCAAATGAGCCCAT 31809 54 P2/PCR TATCCTGAACCAACTGACCTAT 33 618 55 P1/PCRX₁-TTGACACATAAACGAACTG 33 763 56 PO CAGATGGACAAGCACAACC 33 694 57 P2/PCRTCCTGAACCAACTGACCTAT 33 620 58 P1/PCR X₁-CCCATAAGTAGTTGCTGTAT 33 807 59PO GGACAAGCACAACCAGCCACAGC 33 699 60 MB X₂-arm₁-GGACAAGCACAACCAGCCACAGC-33 699 arm₂-X₃ 61 P2/PCR GACCTTTGTGTCCTCAAGAA 33 431 62 P1/PCRX₁-AGGTCAGTTGGTTCAGGATA 33 618 63 PO AGAAACTGCACTGTGACGTGT 33 543 64P2/PCR ATTACAGCGGAGTGAGGTAT 35 217 65 P1/PCR X₁-GTCTTTGCTTTTCAACTGGA 35442 66 PO ATAGAGAAGGCCAGCCATAT 35 270 67 P2/PCR TCAGAGGAGGAGGAAGATACTA35 655 68 P1/PCR X₁-GATTATGCTCTCTGTGAACA 35 844 69 P2/PCRCCCGAGGCAACTGACCTATA 35 610 70 P1/PCR X₁-GTCAATGTGTGTGCTCTGTA 35 770 71PO GACAAGCAAAACCAGACACCTCCAA 35 692 72 PO GACAAGCAAAACCAGACACC 35 692 73P2/PCR TTGTGTGAGGTGCTGGAAGAAT 52 144 74 P1/PCR X₁-CCCTCTCTTCTAATGTTT 52358 75 PO GTGCCTACGCTTTTTATCTA 52 296 76 P2/PCR GTGCCTACGCTTTTTATCTA 52296 77 P1/PCR X₁-GGGGTCTCCAACACTCTGAACA 52 507 78 PO TGCAAACAAGCGATTTCA52 461 79 P2/PCR TCAGGCGTTGGAGACATC 58 157 80 P1/PCRX₁-AGCAATCGTAAGCACACT 58 301 81 P2/PCR TCTGTGCATGAAATCGAA 58 173 82P1/PCR X₁-AGCACACTTTACATACTG 58 291 83 PO TGAAATGCGTTGAATGCA 58 192 84PO TTGCAGCGATCTGAGGTATATG 58 218 85 P2/PCR TACACTGCTGGACAACAT B (11) 51486 P1/PCR X₁-TCATCTTCTGAGCTGTCT B (11) 619 87 P2/PCRTACACTGCTGGACAACATGCA B (11) 514 88 P1/PCR X₁-GTCACATCCACAGCAACAGGTCA B(11) 693 89 PO GTAGGGTTACATTGCTATGA B (11) 590 90 POGTAGGGTTACATTGCTATGAGC B (11) 590 91 P2/PCR TGACCTGTTGCTGTGGATGTGA B(11) 693 92 P1/PCR X₁-TACCTGAATCGTCCGCCAT B (11) 832 93 POATWGTGTGTCCCATCTGC B (11) 794 94 P2/PCR CATGCCATAAATGTATAGA C (18 295 3945) 95 P1/PCR X₁-CACCGCAGGCACCTTATTAA C (18 408 39 45 96 POAGAATTAGAGAATTAAGA C (18 324 39 45 97 P2/PCR GCAGACGACCACTACAGCAAA 39210 98 P1/PCR X₁-ACACCGAGTCCGAGTAATA 39 344 99 PO ATAGGGACGGGGAACCACT 39273 100 P2/PCR TATTACTCGGACTCGGTGT 39 344 101 P1/PCRX₁-CTTGGGTTTCTCTTCGTGTTA 39 558 102 PO GGACCACAAAACGGGAGGAC 39 531 103P2/PCR GAAATAGATGAACCCGACCA 39 703 104 P1/PCR X₁-GCACACCACGGACACACAAA 39886 105 PO TAGCCAGACGGGATGAACCACAGC 39 749 106 P2/PCRAACCATTGAACCCAGCAGAAA 45 430 107 P1/PCR X₁-TCTTTCTTGCCGTGCCTGGTCA 45 527108 PO GTACCGAGGGCAGTGTAATA 45 500 109 P2/PCR AACCATTGAACCCAGCAGAAA 45430 110 P1/PCR X₁-TCTTTCTTGCCGTGCCTGGTCA 45 527 111 P2/PCRGAAACCATTGAACCCAGCAGAAAA 45 428 112 P1/PCR X₁-TTGCTATACTTGTGTTTCCCTACG45 558 113 PO GTACCGAGGGCAGTGTAATA 45 500 114 PO GGACAAACGAAGATTTCACA 45467 115 P2/PCR GTTGACCTGTTGTGTTACCAGCAAT 45 656 116 P1/PCRX₁-CACCACGGACACACAAAGGACAAG 45 868 117 P2/PCR CTGTTGACCTGTTGTGTTACGA 45654 118 P1/PCR X₁-CCACGGACACACAAAGGACAAG 45 868 119 P2/PCRGTTGACCTGTTGTGTTACGA 45 656 120 P1/PCR X₁-ACGGACACACAAAGGACAAG 45 868121 PO GAGTCAGAGGAGGAAAACGATG 45 686 122 PO AGGAAAACGATGAAGCAGATGGAGT 45696 123 PO ACAACTACCAGCCCGACGAGCCGAA 45 730 124 P2/PCRGGAGGAGGATGAAGTAGATA 51 658 125 P1/PCR X₁-GCCCATTAACATCTGCTGTA 51 807126 P2/PCR AGAGGAGGAGGATGAAGTAGATA 51 655 127 P1/PCRX₁-ACGGGCAAACCAGGCTTAGT 51 829 128 PO GCAGGTGTTCAAGTGTAGTA 51 747 129 POTGGCAGTGGAAAGCAGTGGAGACA 51 771 130 P2/PCR TTGGGGTGCTGGAGACAAACATCT 56519 131 P1/PCR X₁-TTCATCCTCATCCTCATCCTCTGA 56 665 132 P2/PCRTGGGGTGCTGGAGACAAACATC 56 520 133 P1/PCR X₁-CATCCTCATCCTCATCCTCTGA 56665 134 P2/PCR TTGGGGTGCTGGAGACAAACAT 56 519 135 P1/PCRX₁-CCACAAACTTACACTCACAACA 56 764 136 PO AAAGTACCAACGCTGCAAGACGT 56 581137 PO AGAACTAACACCTCAAACAGAAAT 56 610 138 PO AGTACCAACGCTGCAAGACGTT 56583 139 P1/PCR X₁-TTGGACAGCTCAGAGGATGAGG 56 656 140 P2/PCRGATTTTCCTTATGCAGTGTG 56 279 141 P1/PCR X₁-GACATCTGTAGCACCTTATT 56 410142 PO GACTATTCAGTGTATGGAGC 56 348 143 PO CAACTGAYCTMYACTGTTATGA A (1631 35) 144 MB X₂-arm₁-CAACTGAYCTMYACTGTTATGA- A (16 arm₂-X₃ 31 35) 145PO GAAMCAACTGACCTAYWCTGCTAT A (33 52 58) 146 MBX₂-arm₁-GAAMCAACTGACCTAYWCTGCTAT- A (33 arm₂-X₃ 52 58) 147 POAAGACATTATTCAGACTC C (18 45 39) 148 MBX₂-arm₁-AAGACATTATTCAGACTC-arm₂-X₃ C (18 45 39)

TABLE 2 L1-specific sequences for inclusion in NASBA/PCR primers andprobes Primer/probe SEQ ID type Sequence 149 P2/PCR AATGGCATTTGTTGGGGTAA150 P1/PCR X₁-TCATATTCCTCCCCATGTC 151 PO TTGTTACTGTTGTTGATACTAC 152P2/PCR AATGGCATTTGTTGGGRHAA 153 P1/PCR X₁-TCATATTCCTCMMCATGDC 154 POTTGTTACTGTTGTTGATACYAC 155 PO TTGTTACTGTTGTTGATACCAC 156 P2/PCRAATGGCATTTGTTGGSIIAA 157 P2/PCR AATGGCATTTGTTGGIIHAA 158 P2/PCRAATGGCATTTGTTGGIRIAA 159 P2/PCR AATGGCATTTGTTGGGGTAA 160 P2/PCRAATGGCATTTGTTGGGGAAA 161 P2/PCR AATGGCATTTGTTGGCATAA 162 P2/PCRAATGGCATTTGTTGGGGCAA 163 P2/PCR AATGGCATTTGTTGGCACAA 164 P1/PCRX₁-TCATATTCCTCMICATGIC 165 P1/PCR X₁-TCATATTCCTCAACATGIC 166 P1/PCRX₁-TCATATTCCTCIICATGTC 167 P1/PCR X₁-TCATATTCCTCIICATGGC 168 P1/PCRX₁-TCATATTCCTCIICATGAC 3′ 169 P1/PCR X₁-TCATATTCCTCIICATGCC 3′

Preferred primers suitable for use in detection of HPV L1 and E6 mRNA byNASBA are listed in the following tables. However, these are merelyillustrative and it is not intended that the scope of the inventionshould be limited to these specific molecules.

In the following Tables the NASBA P2 primers (p2) include the sequenceGATGCAAGGTCGCATATGAG (SEQ ID No. 170) at the 5′ end; the NASBA P1primers (p1) include the sequence AATTCTAATACGACTCACTATAGGGAGAAGG (SEQID No. 172) at the 5′ end. Oligonucleotides suitable for use as probesare identified by “po”. The P2 primers generally contain HPV sequencesfrom the positive strand, whereas the P1 primers generally contain HPVsequences from the negative strand. nt-refers to nucleotide position inthe relevant HPV genomic sequence.

TABLE 3 Preferred E6 NASBA primers and probes HPV Primer name SequenceType nt HAe6701p2 GATGCAAGGTCGCATATGAGCCACAGGAGCGACCC 16 116 (SEQ ID173) AGAAAGTTA HAe6701p1 AATTCTAATACGACTCACTATAGGGAGAAGGACGG 16 368 (SEQID 174) TTTGTTGTATTGCTGTTC HAe6702p2 GATGCAAGGTCGCATATGAGCCACAGGAGCGACCC16 116 (SEQ ID 175) AGAAA HAe6702p1 AATTCTAATACGACTCACTATAGGGAGAAGGGGTT16 368 (SEQ ID 176) TGTTGTATTGCTGTTC HPV16p1AATTCTAATACGACTCACTATAGGGAGAAGGATTC 16 258 (SEQ ID 177) CCATCTCTATATACTAHAe6702Ap1 AATTCTAATACGACTCACTATAGGGAGAAGGTCA 16 208 (SEQ ID 178)CGTCGCAGTAACTGT HAe6702Bp1 AATTCTAATACGACTCACTATAGGGAGAAGGTTG 16 191(SEQ ID 179) CTTGCAGTACACACA HAe6702Cp1AATTCTAATACGACTCACTATAGGGAGAAGGTGC 16 186 (SEQ ID 180) AGIACACACATTCTAHAe6702Dp1 AATTCTAATACGACTCACTATAGGGAGAAGGGCA 16 185 (SEQ ID 181)GTACACACATTCTAA H16e6702Ap2 GATGCAAGGTCGCATATGAGACAGTTATGCACAGA 16 142(SEQ ID 182) GCT H16e6702Bp2 GATGCAAGGTCGCATATGAGATATTAGAATGTGTG 16 182(SEQ ID 183) TAC H16e6702Cp2 GATGCAAGGTCGCATATGAGTTAGAATGTGTGTAC 16 185(SEQ ID 184) TGC H16e6702Dp2 GATGCAAGGTCGCATATGAGGAATGTGTGTACTGC 16 188(SEQ ID 185) AAG H16e6702Apo ACAGTTATGCACAGAGCT 16 142 (SEQ ID 10)H16e6702Bpo ATATTAGAATGTGTGTAC 16 182 (SEQ ID 11) H16e6702CpoTTAGAATGTGTGTACTGC 16 185 (SEQ ID 12) H16e6702Dpo GAATGTGTGTACTGCAAG 16188 (SEQ ID 13) HAe6701po CTTTGCTTTTCGGGATTTATGC 16 235 (SEQ ID 18)HAe6702po TATGACTTTGCTTTTCGGGA 16 230 (SEQ ID 19) HAe6702mb1X₂-cgcatgTATGACTTTGCTTTTCGGGAcatgcg- 16 230 (SEQ ID 186) X₃ HAe6702mb2X₂-ccagctTATGACTTTGCTTTTCGGGAagctgg- 16 230 (SEQ ID 187) X₃ HAe6702mb3X₂-cacgcTATGACTTTGCTTTTCGGGAgcgtg-X₃ 16 230 (SEQ ID 188) H16e6702mb4X₂-cgatcgTATGACTTTGCTTTTCGGGAcgatcg- 16 230 (SEQ ID 189) X₃ HAe6703p2GATGCAAGGTCGCATATGAGCAGAGGAGGAGGATG 16 656 (SEQ ID 190) AAATAGTAHAe6703p1 AATTCTAATACGACTCACTATAGGGAGAAGGGCAC 16 741 (SEQ ID 191)AACCGAAGCGTAGAGTCACAC HAe6703po TGGACAAGCAGAACCGGACAGAGC 16 687 (SEQ ID23) HAe6704p2 GATGCAAGGTCGCATATGAGCAGAGGAGGAGGATG 16 656 (SEQ ID 192)AAATAGA HAe6704p1 AATTCTAATACGACTCACTATAGGGAGAAGGGCAC 16 741 (SEQ ID193) AACCGAAGCGTAGAGTCA HAe6704po AGCAGAACCGGACAGAGCCCATTA 16 693 (SEQID 26) H18e6701p2 GATGCAAGGTCGCATATGAGACGATGAAATAGATG 18 702 (SEQ ID194) GAGTT H18e6701p1 AATTCTAATACGACTCACTATAGGGAGAAGGCACG 18 869 (SEQ ID195) GACACACAAAGGACAG Hl8e6701po AGCCGAACCACAACGTCACA 18 748 (SEQ ID 29)H18e6702p2 GATGCAAGGTCGCATATGAGGAAAACGATGAAATA 18 698 (SEQ ID 196)GATGGAG H18e6702p1 AATTCTAATACGACTCACTATAGGGAGAAGGACAC 18 869 (SEQ ID197) CACGGACACACAAAGGACAG H18e6702po GAACCACAACGTCACACAATG 18 752 (SEQID 32) H18e6702mb1 X₂-cgcatgGAACCACAACGTCACACAATGcatgcg- 18 752 (SEQ ID198) X₃ H18e6702mb2 X₂-ccgtcgGAACCACAACGTCACACAATGcgacgg- 18 752 (SEQ ID199) X₃ H18e6702mb3 X₂-cggaccGAACCACAACGTCACACAATGggtccg- 18 752 (SEQ ID200) X₃ H18e6702mb4 X₂-cgatcgGAACCACAACGTCACACAATGcgatcg- 18 752 (SEQ ID201) X₃ H18e6703p2 GATGCAAGGTCGCATATGAGTTCCGGTTGACCTTC 18 651 (SEQ ID202) TATGT H18e6703p1 AATTCTAATACGACTCACTATAGGGAGAAGGGGTC 18 817 (SEQ ID203) GTCTGCTGAGCTTTCT H18e6704p2 GATGCAAGGTCGCATATGAGGCAAGACATAGAAAT 18179 (SEQ ID 204) AACCTG H18e6704p1 AATTCTAATACGACTCACTATAGGGAGAAGGACCC18 379 (SEQ ID 205) AGTGTTAGTTAGTT H18e6704po TGCAAGACAGTATTGGAACT 18207 (SEQ ID 38) H31e6701p2 GATGCAAGGTCGCATATGAGGGAAATACCCTACGA 31 164(SEQ ID 206) TGAAC H31e6701p1 AATTCTAATACGACTCACTATAGGGAGAAGGGGAC 31 423(SEQ ID 207) ACAACGGTCTTTGACA H31e6701po ATAGGGACGACACACCACACGGAG 31 268(SEQ ID 41) H31e6702p2 GATGCAAGGTCGCATATGAGGGAAATACCCTACGA 31 164 (SEQID 208) TGAACTA H31e6702p1 AATTCTAATACGACTCACTATAGGGAGAAGGCTGG 31 423(SEQ ID 209) ACACAACGGTCTTTGACA H31e6702po TAGGGACGACACACCACACGGA 31 269(SEQ ID 44) H31e6703p2 GATGCAAGGTCGCATATGAGACTGACCTCCACTGT 31 617 (SEQID 210) TATGA H31e6703p1 AATTCTAATACGACTCACTATAGGGAGAAGGTATC 31 766 (SEQID 211) TACTTGTGTGCTCTGT H31e6703po GACAAGCAGAACCGGACACATC 31 687 (SEQID 47) H31e6704p2 GATGCAAGGTCGCATATGAGTGACCTCCACTGTTA 31 619 (SEQ ID212) TGAGCAATT H31e6704p1 AATTCTAATACGACTCACTATAGGGAGAAGGTGCG 31 766(SEQ ID 213) AATATCTACTTGTGTGCTCT GT H31e6704poGGACAAGCAGAACCGGACACATCCAA 31 686 (SEQ ID 50) H31e6704mb1X₂-ccgaaggGGACAAGCAGAACCGGACACATCC 31 686 (SEQ ID 214) AAccttcgg-X₃H31e6704mb2 X₂-ccgtcgGGACAAGCAGAACCGGACACATCCA 31 686 (SEQ ID 215)Acgacgg-X₃ H31e6704mb3 X₂-cacgtcgGGACAAGCAGAACCGGACACATCCAA 31 686 (SEQID 216) cgacgtg-X₃ H31e6704mb4 X₂-cgcagcGGACAAGCAGAACCGGACACATCCAA 31686 (SEQ ID 217) gctgcg-X₃ H31e6704mb5X₂-cgatcgGGACAAGCAGAACCGGACACATCCAA 31 686 (SEQ ID 218) cgatcg-X₃H31e6705p2 GATGCAAGGTCGCATATGAGACTGACCTCCACTGT 31 617 (SEQ ID 219) TATH31e6705p1 AATTCTAATACGACTCACTATAGGGAGAAGGCACG 31 809 (SEQ ID 220)ATTCCAAATGAGCCCAT H33e6701p2 GATGCAAGGTCGCATATGAGTATCCTGAACCAACT 33 618(SEQ ID 221) GACCTAT H33e6701p1 AATTCTAATACGACTCACTATAGGGAGAAGGTTGA 33763 (SEQ ID 222) CACATAAACGAACTG H33e6701po CAGATGGACAAGCACAACC 33 694(SEQ ID 56) H33e6703p2 GATGCAAGGTCGCATATGAGTCCTGAACCAACTGA 33 620 (SEQID 223) CCTAT H33e6703p1 AATTCTAATACGACTCACTATAGGGAGAAGGCCCA 33 807 (SEQID 224) TAAGTAGTTGCTGTAT H33e6703po GGACAAGCACAACCAGCCACAGC 33 699 (SEQID 59) H33e6703mb1 X₂-ccaagcGGACAAGCACAACCAGCCACAGCgct 33 699 (SEQ ID225) tgg-X₃ H33e6703mb2 X₂-ccaagcgGGACAAGCACAACCAGCCACAGC 33 699 (SEQ ID226) cgcttgg-X₃ H33e6703mb3 X₂-cccagcGGACAAGCACAACCAGCCACAGCgct 33 699(SEQ ID 227) ggg-X₃ H33e6703mb4 X₂-ccaaagcGGACAAGCACAACCAGCCACAGCg 33699 (SEQ ID 228) ctttgg-X₃ H33e6703mb5X₂-cctgcGGACAAGCACAACCAGCCACAGCgcagg- 33 699 (SEQ ID 229) X₃ H33e6703mb6X₂-ccgatcgGGACAAGCACAACCAGCCACAGCcga 33 699 (SEQ ID 230) tcg-X₃H33e6702p2 GATGCAAGGTCGCATATGAGGACCTTTGTGTCCTC 33 431 (SEQ ID 231) AAGAAH33e6702p1 AATTCTAATACGACTCACTATAGGGAGAAGGAGGT 33 618 (SEQ ID 232)CAGTTGGTTCAGGATA H33e6702po AGAAACTGCACTGTGACGTGT 33 543 (SEQ ID 63)H35e6701p2 GATGCAAGGTCGCATATGAGATTACAGCGGAGTGA 35 217 (SEQ ID 233) GGTATH35e6701p1 AATTCTAATACGACTCACTATAGGGAGAAGGGTCT 35 442 (SEQ ID 234)TTGCTTTTCAACTGGA H35e5601po ATAGAGAAGGCCAGCCATAT 35 270 (SEQ ID 66)H35e6702p2 GATGCAAGGTCGCATATGAGTCAGAGGAGGAGGAA 35 655 (SEQ ID 235)GATACTA H35e6702p1 AATTCTAATACGACTCACTATAGGGAGAAGGGATT 35 844 (SEQ ID236) ATGCTCTCTGTGAACA H35e6703p2 GATGCAAGGTCGCATATGAGCCCGAGGCAACTGAC 35610 (SEQ ID 237) CTATA H35e6703p1 AATTCTAATACGACTCACTATAGGGAGAAGGGTCA 35770 (SEQ ID 238) ATGTGTGTGCTCTGTA H35e6702po GACAAGCAAAACCAGACACCTCCAA35 692 (SEQ ID 71) H35e6703po GACAAGCAAAACCAGACACC 35 692 (SEQ ID 72)H52e6701p2 GATGCAAGGTCGCATATGAGTTGTGTGAGGTGCTG 52 144 (SEQ ID 239)GAAGAAT H52e6701p1 AATTCTAATACGACTCACTATAGGGAGAAGGCCCT 52 358 (SEQ ID240) CTCTTCTAATGTTT H52e6701po GTGCCTACGCTTTTTATCTA 52 296 (SEQ ID 75)H52e6702p2 GATGCAAGGTCGCATATGAGGTGCCTACGCTTTTT 52 296 (SEQ ID 241) ATCTAH52e6702p1 AATTCTAATACGACTCACTATAGGGAGAAGGGGGG 52 507 (SEQ ID 242)TCTCCAACACTCTGAACA H52e6702po TGCAAACAAGCGATTTCA 52 461 (SEQ ID 78)H58e6701p2 GATGCAAGGTCGCATATGAGTCAGGCGTTGGAGAC 58 157 (SEQ ID 243) ATCH58e6701p1 AATTCTAATACGACTCACTATAGGGAGAAGGAGCA 58 301 (SEQ ID 244)ATCGTAAGCACACT H58e6702p2 GATGCAAGGTCGCATATGAGTCTGTGCATGAAATC 58 173(SEQ ID 245) GAA H58e6702p1 AATTCTAATACGACTCACTATAGGGAGAAGGAGCA 58 291(SEQ ID 246) CACTTTACATACTG H58e6701po TGAAATGCGTTGAATGCA 58 192 (SEQ ID83) H58e6702po TTGCAGCGATCTGAGGTATATG 58 218 (SEQ ID 84) HBe6701p2GATGCAAGGTCGCATATGAGTACACTGCTGGACAA B (11) 514 (SEQ ID 247) CATHBe6701p1 AATTCTAATACGACTCACTATAGGGAGAAGGTCAT B (11) 619 (SEQ ID 248)CTTCTGAGCTGTCT HBe6702p2 GATGCAAGGTCGCATATGAGTACACTGCTGGACAA B (11) 514(SEQ ID 249) CATGCA HBe6702p1 AATTCTAATACGACTCACTATAGGGAGAAGGGTCA B (11)693 (SEQ ID 250) CATCCACAGCAACAGGTCA HBe6701po GTAGGGTTACATTGCTATGA B(11) 590 (SEQ ID 89) HBe6702po GTAGGGTTACATTGCTATGAGC B (11) 590 (SEQ ID90) HBe6703p2 GATGCAAGGTCGCATATGAGTGACCTGTTGCTGTG B (11) 693 (SEQ ID251) GATGTGA HBe6703p1 AATTCTAATACGACTCACTATAGGGAGAAGGTACC B (11) 832(SEQ ID 252) TGAATCGTCCGCCAT HBe6703po ATWGTGTGTCCCATCTGC B (11) 794(SEQ ID 93) HCe6701p2 GATGCAAGGTCGCATATGAGCATGCCATAAATGTA C (18 295 (SEQID 253) TAGA 39 45) HCe6701p1 AATTCTAATACGACTCACTATAGGGAGAAGGCACC C (18408 (SEQ ID 254) GCAGGCACCTTATTAA 39 45 HCe6701po AGAATTAGAGAATTAAGA C(18 324 (SEQ ID 96) 39 45 H39e6701p2 GATGCAAGGTCGCATATGAGGCAGACGACCACTAC39 210 (SEQ ID 255) AGCAAA H39e6701p1AATTCTAATACGACTCACTATAGGGAGAAGGACAC 39 344 (SEQ ID 256) CGAGTCCGAGTAATAH39e6701po ATAGGGACGGGGAACCACT 39 273 (SEQ ID 99) H39e6702p2GATGCAAGGTCGCATATGAGTATTACTCGGACTCG 39 344 (SEQ ID 257) GTGT H39e6702p1AATTCTAATACGACTCACTATAGGGAGAAGGCTTG 39 558 (SEQ ID 258)GGTTTCTCTTCGTGTTA H39e6702po GGACCACAAAACGGGAGGAC 39 531 (SEQ ID 102)H39e6703p2 GATGCAAGGTCGCATATGAGGAAATAGATGAACCC 39 703 (SEQ ID 259) GACCAH39e6703p1 AATTCTAATACGACTCACTATAGGGAGAAGGGCAC 39 886 (SEQ ID 260)ACCACGGACACACAAA H39e6703po TAGCCAGACGGGATGAACCACAGC 39 749 (SEQ ID 105)HPV45p2 GATGCAAGGTCGCATATGAGAACCATTGAACCCAG 45 430 (SEQ ID 261) CAGAAAHPV45p1 AATTCTAATACGACTCACTATAGGGAGAAGGTCTT 45 527 (SEQ ID 262)TCTTGCCGTGCCTGGTCA HPV45po GTACCGAGGGCAGTGTAATA 45 500 (SEQ ID 108)H45e6701p2 GATGCAAGGTCGCATATGAGAACCATTGAACCCAG 45 430 (SEQ ID 263)CAGAAA H45e6701p1 AATTCTAATACGACTCACTATAGGGAGAAGGTCTT 45 527 (SEQ ID264) TCTTGCCGTGCCTGGTCA H45e6702p2 GATGCAAGGTCGCATATGAGGAAACCATTGAACCC45 428 (SEQ ID 265) AGCAGAAAA H45e6702p1AATTCTAATACGACTCACTATAGGGAGAAGGTTGC 45 558 (SEQ ID 266)TATACTTGTGTTTCCCTACG H45e6701po GTACCGAGGGCAGTGTAATA 45 500 (SEQ ID 113)H45e6702po GGACAAACGAAGATTTCACA 45 467 (SEQ ID 114) H45e6703p2GATGCAAGGTCGCATATGAGGTTGACCTGTTGTGT 45 656 (SEQ ID 267) TACCAGCAATH45e6703p1 AATTCTAATACGACTCACTATAGGGAGAAGGCACC 45 868 (SEQ ID 268)ACGGACACACAAAGGACAAG H45e6704p2 GATGCAAGGTCGCATATGAGCTGTTGACCTGTTGT 45654 (SEQ ID 269) GTTACGA H45e6704p1 AATTCTAATACGACTCACTATAGGGAGAAGGCCAC45 868 (SEQ ID 270) GGACACACAAAGGACAAG H45e6705p2GATGCAAGGTCGCATATGAGGTTGACCTGTTGTGT 45 656 (SEQ ID 271) TACGA H45e6705p1AATTCTAATACGACTCACTATAGGGAGAAGGACGG 45 868 (SEQ ID 272) ACACACAAAGGACAAGH45e6703po GAGTCAGAGGAGGAAAACGATG 45 686 (SEQ ID 121) H45e6704poAGGAAAACGATGAAGCAGATGGAGT 45 696 (SEQ ID 122) H45e6705poACAACTACCAGCCCGACGAGCCGAA 45 730 (SEQ ID 123) H51e6701p2GATGCAAGGTCGCATATGAGGGAGGAGGATGAAGT 51 658 (SEQ ID 273) AGATA H51e6701p1AATTCTAATACGACTCACTATAGGGAGAAGGGCCC 51 807 (SEQ ID 274) ATTAACATCTGCTGTAH51e6702p2 GATGCAAGGTCGCATATGAGAGAGGAGGAGGATGA 51 655 (SEQ ID 275)AGTAGATA H51e6702p1 AATTCTAATACGACTCACTATAGGGAGAAGGACGG 51 829 (SEQ ID276) GCAAACCAGGCTTAGT H51e6701po GCAGGTGTTCAAGTGTAGTA 51 747 (SEQ ID128) H51e6702po TGGCAGTGGAAAGCAGTGGAGACA 51 771 (SEQ ID 129) H56e6701p2GATGCAAGGTCGCATATGAGTTGGGGTGCTGGAGA 56 519 (SEQ ID 277) CAAACATCTH56e6701p1 AATTCTAATACGACTCACTATAGGGAGAAGGTTCA 56 665 (SEQ ID 278)TCCTCATCCTCATCCTCTGA H56e6702p2 GATGCAAGGTCGCATATGAGTGGGGTGCTGGAGAC 56520 (SEQ ID 279) AAACATC H56e6702p1 AATTCTAATACGACTCACTATAGGGAGAAGGCATC56 665 (SEQ ID 280) CTCATCCTCATCCTCTGA H56e6703p2GATGCAAGGTCGCATATGAGTTGGGGTGCTGGAGA 56 519 (SEQ ID 281) CAAACATH56e6703p1 AATTCTAATACGACTCACTATAGGGAGAAGGCCAC 56 764 (SEQ ID 282)AAACTTACACTCACAACA H56e6701po AAAGTACCAACGCTGCAAGACGT 56 581 (SEQ ID136) H56e6702po AGAACTAACACCTCAAACAGAAAT 56 610 (SEQ ID 137) H56e6703poAGTACCAACGCTGCAAGACGTT 56 583 (SEQ ID 138) H56e6703po1TTGGACAGCTCAGAGGATGAGG 56 656 (SEQ ID 139) H56e6704p2GATGCAAGGTCGCATATGAGGATTTTCCTTATGCA 56 279 (SEQ ID 283) GTGTG H56e6704p1AATTCTAATACGACTCACTATAGGGAGAAGGGACA 56 410 (SEQ ID 284) TCTGTAGCACCTTATTH56e6704po GACTATTCAGTGTATGGAGC 56 348 (SEQ ID 142) HPVAPO1ACAACTGAYCTMYACTGTTATGA A (16 (SEQ ID 143) 31 35) HPVApo1Amb1X₂-cgcatgCAACTGAYCTMYACTGTTATGAcatgc A (16 (SEQ ID 285) g-X₃ 31 35)HPVApo1Amb2 X₂-ccgtcgCAACTGAYCTMYACTGTTATGAcga A (16 (SEQ ID 286) cgg-X₃31 35) HPVApo1Amb3 X₂-ccacccCAACTGAYCTMYACTGTTATGAgg A (16 (SEQ ID 287)gtgg-X₃ 31 35) HPVApo1Amb4 X₂-cgatcgCAACTGAYCTMYACTGTTATGAcga A (16 (SEQID 288) tcg-X₃ 31 35) HPVAPO4A GAAMCAACTGACCTAYWCTGCTAT A (33 (SEQ ID145) 52 58) HPVAPO4Amb1 X₂-ccaagcGAAMCAACTGACCTAYWCTGCTATgc A (33 (SEQID 289) ttgg-X₃ 52 58) HPVAPO4Amb2 X₂-ccaagccGAAMCAACTGACCTAYWCTGCTAT A(33 (SEQ ID 290) ggcttgg-X₃ 52 58) HPVAPO4Amb3X₂-ccaagcgGAAMCAACTGACCTAYWCTGCTA A (33 (SEQ ID 291) Tcgcttgg-X₃ 52 58)HPVAPO4Amb4 X₂-ccagcgGAAMCAACTGACCTAYWCTGCTATcg A (33 (SEQ ID 292)ctgg-X₃ 52 58) HPVAPO4Amb5 X₂-cgatcgGAAMCAACTGACCTAYWCTGCTATcg A (33(SEQ ID 293) atcg-X₃ 52 58) HPVCPO4 AAGACATTATTCAGACTC C (18 (SEQ ID147) 45 39) HPVCPO4Amb1 X₂-ccaagcAAGACATTATTCAGACTCgcttgg-X₃ C (18 (SEQID 294) 45 39) HPVCPO4Amb2 X₂-cgcatgAAGACATTATTCAGACTCcatgcg-X₃ C (18(SEQ ID 295) 45 39) HPVCPO4Amb3 X₂-cccagcAAGACATTATTCAGACTCgctggg-X₃ C(18 (SEQ ID 296) 45 39) HPVCPO4Amb4 X₂-cgatcgAAGACATTATTCAGACTCcgatcg-X₃C (18 (SEQ ID 297) 45 39)Pairs of P1 and P2 primers having the same prefix (e.g. HAe6701p1 andHAe6701p2) are intended to be used in combination. However, othercombinations may also be used, as summarised below for HPV types 16, 18,31, 33 and 45.Suitable primer-pairs for amplification of HPV 16 E6 mRNA are asfollows:HAe6701p2 or HAe6702p2 (both nt 116) with HAe6701p1 or HAe6702p1 (bothnt 368).HAe6701p2 or HAe6702p2 (both nt 116) with HPV16p1(nt 258).H16e6702Ap2 (nt 142), H16e6702 Bp2 (nt 182), H16e6702 Cp2 (nt 185) orH16e6702Dp2 (nt 188) with HAe6701p1 or HAe6702p1 (both nt 368).HAe6701p2 or HAe6702p2 (both nt 116) with HAe6702Ap1 (nt 208), HAe6702Bp1 (nt 191), HAe6702 Cp1 (nt 186) or HAe6702Dp1 (185). Thesecombinations are suitable for amplification of all E6 splice variants.HAe6703p2 or HAe6704p2 (both nt 656) with HAe6703p1 or HAe6704p1 (bothnt 741). These combinations are suitable for amplification of alltranscripts containing the E7 coding region (at least up to nt 741).The following primer-pairs are preferred for amplification of HPV 18 E6mRNA:H18e6701p2 (nt 702) or H18e6702p2 (nt 698) with H18e6701p1 or H18e6702p1(both nt 869).H18e6703p2 (nt 651) with H18e6703p1 (nt 817). H18e6704p2 (nt 179) withH18e6704p1 (nt 379).The following primer-pairs are preferred for amplification of HPV 31 E6mRNA:H31e6701p2 or H31e6702p2 (both nt 164) with H31e6701p1 or H31e6702p1(both nt 423).H31e6703p2 (nt 617), H31e6704p2 (nt 619) or H31e6705p2 (nt 617) withH31e6703p1 (nt 766), H31e6704p1 (766) or H31e6705p1 (nt 809).The following primer-pairs are preferred for amplification of HPV 33 E6mRNA:H33e6701p2 (nt 618) or H33e6703p2 (nt 620) with H33e6701p1 (nt 763) orH33e6703p1 (nt 807).H33e6702p2 (nt 431) with H33e6702p1 (nt 618).The following primer pair is preferred for amplification of HPV 45:HPV45p2 (nt 430) with HPV45p1(nt 527)

TABLE 4 E6 PCR primers HPV Primer name Sequence type nt HAe6701PCR2 (SEQID 1) CCACAGGAGCGACCCAGAAAGTTA 16 116 HAe6701PCR1 (SEQ ID 2)ACGGTTTGTTGTATTGCTGTTC 16 368 HAe6702PCR2 (SEQ ID 3)CCACAGGAGCGACCCAGAAA 16 116 HAe6702PCR1 (SEQ ID 4) GGTTTGTTGTATTGCTGTTC16 368 HAe6703PCR2 (SEQ ID 21) CAGAGGAGGAGGATGAAATAGTA 16 656HAe6703PCR1 (SEQ ID 22) GCACAACCGAAGCGTAGAGTCACAC 16 741 HAe6704PCR2(SEQ ID 24) CAGAGGAGGAGGATGAAATAGA 16 656 HAe6704PCR1 (SEQ ID 25)GCACAACCGAAGCGTAGAGTCA 16 741 H18e6701PCR2 (SEQ ID 27)ACGATGAAATAGATGGAGTT 18 702 H18e6701PCR1 (SEQ ID 28)CACGGACACACAAAGGACAG 18 869 H18e6702PCR2 (SEQ ID 30)GAAAACGATGAAATAGATGGAG 18 698 H18e6702PCR1 (SEQ ID 31)ACACCACGGACACACAAAGGACAG 18 869 H18e6703PCR2 (SEQ ID 34)TTCCGGTTGACCTTCTATGT 18 651 H18e6703PCR1 (SEQ ID 35)GGTCGTCTGCTGAGCTTTCT 18 817 H18e6704PCR2 (SEQ ID 36)GCAAGACATAGAAATAACCTG 18 179 H18e6704PCR1 (SEQ ID 37) ACCCAGTGTTAGTTAGTT18 379 H31e6701PCR2 (SEQ ID 39) GGAAATACCCTACGATGAAC 31 164 H31e6701PCR1(SEQ ID 40) GGACACAACGGTCTTTGACA 31 423 H31e6702PCR2 (SEQ ID 42)GGAAATACCCTACGATGAACTA 31 164 H31e6702PCR1 (SEQ ID 43)CTGGACACAACGGTCTTTGACA 31 423 H31e6703PCR2 (SEQ ID 45)ACTGACCTCCACTGTTATGA 31 617 H31e6703PCR1 (SEQ ID 46)TATCTACTTGTGTGCTCTGT 31 766 H31e6704PCR2 (SEQ ID 48)TGACCTCCACTGTTATGAGCAATT 31 619 H31e6704PCR1 (SEQ ID 49)TGCGAATATCTACTTGTGTGCTCTGT 31 766 H31e6705PCR2 (SEQ ID 52)ACTGACCTCCACTGTTAT 31 617 H31e6705PCR1 (SEQ ID 53) CACGATTCCAAATGAGCCCAT31 809 H33e6701PCR2 (SEQ ID 54) TATCCTGAACCAACTGACCTAT 33 618H33e6701PCR1 (SEQ ID 55) TTGACACATAAACGAACTG 33 763 H33e6703PCR2 (SEQ ID57) TCCTGAACCAACTGACCTAT 33 620 H33e6703PCR1 (SEQ ID 58)CCCATAAGTAGTTGCTGTAT 33 807 H33e6702PCR2 (SEQ ID 61)GACCTTTGTGTCCTCAAGAA 33 431 H33e6702PCR1 (SEQ ID 62)AGGICAGTIGGITCAGGATA 33 618 H35e6701PCR2 (SEQ ID 64)ATTACAGCGGAGTGAGGTAT 35 217 H35e6701PCR1 (SEQ ID 65)GTCTTTGCTTTTCAACTGGA 35 442 H35e6702PCR2 (SEQ ID 67)TCAGAGGAGGAGGAAGATACTA 35 655 H35e6702PCR1 (SEQ ID 68)GATTATGCTCTCTGTGAACA 35 844 H35e6703PCR2 (SEQ ID 69)CCCGAGGCAACTGACCTATA 35 610 H35e6703PCR1 (SEQ ID 70)GTCAATGTGTGTGCTCTGTA 35 770 H52e6701PCR2 (SEQ ID 73)TTGTGTGAGGTGCTGGAAGAAT 52 144 H52e6701PCR1 (SEQ ID 74)CCCTCTCTTCTAATGTTT 52 358 H52e6702PCR2 (SEQ ID 75) GTGCCTACGCTTTTTATCTA52 296 H52e6702PCR1 (SEQ ID 77) GGGGTCTCCAACACTCTGAACA 52 507H58e6701PCR2 (SEQ ID 79) TCAGGCGTTGGAGACATC 58 157 H58e6701PCR1 (SEQ ID80) AGCAATCGTAAGCACACT 58 301 H58e6702PCR2 (SEQ ID 81)TCTGTGCATGAAATCGAA 58 173 H58e6702PCR1 (SEQ ID 82) AGCACACTTTACATACTG 58291 HBe6701PCR2 (SEQ ID 85) TACACTGCTGGACAACAT B (11) 514 HBe6701PCR1(SEQ ID 86) TCATCTTCTGAGCTGTCT B (11) 619 HBe6702PCR2 (SEQ ID 87)TACACTGCTGGACAACATGCA B (11) 514 HBe6702PCR1 (SEQ ID 88)GTCACATCCACAGCAACAGGTCA B (11) 693 HBe6703PCR2 (SEQ ID 91)TGACCTGTTGCTGTGGATGTGA B (11) 693 HBe6703PCR1 (SEQ ID 92)TACCTGAATCGTCCGCCAT B (11) 832 HCe6701PCR2 (SEQ ID 94)CATGCCATAAATGTATAGA C (18 295 39 45 HCe6701PCR1 (SEQ ID 95)CACCGCAGGCACCTTATTAA C (18 408 39 45 H39e6701PCR2 (SEQ ID 97)GCAGACGACCACTACAGCAAA 39 210 H39e6701PCR1 (SEQ ID 98)ACACCGAGTCCGAGTAATA 39 344 H39e6702PCR2 (SEQ ID 100) TATTACTCGGACTCGGTGT39 344 H39e6702PCR1 (SEQ ID 101) CTTGGGTTTCTCTTCGTGTTA 39 558H39e6703PCR2 (SEQ ID 103) GAAATAGATGAACCCGACCA 39 703 H39e6703PCR1 (SEQID 104) GCACACCACGGACACACAAA 39 886 H45e6701PCR2 (SEQ ID 106)AACCATTGAACCCAGCAGAAA 45 430 H45e6701PCR1 (SEQ ID 107)TCTTTCTTGCCGTGCCTGGTCA 45 527 H45e6702PCR2 (SEQ ID 111)GAAACCATTGAACCCAGCAGAAAA 45 428 H45e6702PCR1 (SEQ ID 112)TTGCTATACTTGTGTTTCCCTACG 45 558 H45e6703PCR2 (SEQ ID 115)GTTGACCTGTTGTGTTACCAGCAAT 45 656 H45e6703PCR1 (SEQ ID 116)CACCACGGACACACAAAGGACAAG 45 868 H45e6704PCR2 (SEQ ID 117)CTGTTGACCTGTTGTGTTACGA 45 654 H4Se6704PCR1 (SEQ ID 118)CCACGGACACACAAAGGACAAG 45 868 H45e6705PCR2 (SEQ ID 119)GTTGACCTGTTGTGTTACGA 45 656 H45e6705PCR1 (SEQ ID 120)ACGGACACACAAAGGACAAG 45 868 H51e6701PCR2 (SEQ ID 124)GGAGGAGGATGAAGTAGATA 51 658 H51e6701PCR1 (SEQ ID 125)GCCCATTAACATCTGCTGTA 51 807 H51e6702PCR2 (SEQ ID 126)AGAGGAGGAGGATGAAGTAGATA 51 655 H51e6702PCR1 (SEQ ID 127)ACGGGCAAACCAGGCTTAGT 51 829 H56e6701PCR2 (SEQ ID 130)TTGGGGTGCTGGAGACAAACATCT 56 519 H56e6701PCR1 (SEQ ID 131)TTCATCCTCATCCTCATCCTCTGA 56 665 H56e6702PCR2 (SEQ ID 132)TGGGGTGCTGGAGACAAACATC 56 520 H56e6702PCR1 (SEQ ID 133)CATCCTCATCCTCATCCTCTGA 56 665 H56e6703PCR2 (SEQ ID 134)TTGGGGTGCTGGAGACAAACAT 56 519 H56e6703PCR1 (SEQ ID 135)CCACAAACTTACACTCACAACA 56 764 H56e6704PCR2 (SEQ ID 140)GATTTTCCTTATGCAGTGTG 56 279 H56e6704PCR1 (SEQ ID 141)GACATCTGTAGCACCTTATT 56 410Preferred PCR primer-pairs for HPV types 16, 18, 31 and 33 are analogousto the NASBA primer-pairs.

TABLE 5 Preferred L1 NASBA primers and probes Primer name SequenceOnc2A2 5′ GATGCAAGGTCGCATATGAGAATGGCATTTGTTG (SEQ ID 298) GGGTAA 3′Onc2A1 5′ AATTCTAATACGACTCACTATAGGGAGAAGGTCA (SEQ ID 299)TATTCCTCCCCATGTC 3′ Onc2PoA 5′ TTGTTACTGTTGTTGATACTAC 3′ (SEQ ID 151)Onc2B2 5′ GATGCAAGGTCGCATATGAGAATGGCATTTGTTG (SEQ ID 300) GSRHAA 3′Onc2B1 5′ AATTCTAATACGACTCACTATAGGGAGAAGGTCA (SEQ ID 301)TATTCCTCMMCATGDC 3′ Onc2PoB 5′ TTGTTACTGTTGTTGATACYAC 3′ (SEQ ID 154)Onc2PoC 5′ TTGTTACTGTTGTTGATACCAC 3′ (SEQ ID 155) Onc2C25′ GATGCAAGGTCGCATATGAGAATGGCATTTGTTG (SEQ ID 302) GSIIAA 3′ Onc2D25′ GATGCAAGGTCGCATATGAGAATGGCATTTGTTG (SEQ ID 303) GIIHAA 3′ Onc2E25′ GATGCAAGGTCGCATATGAGAATGGCATTTGTTG (SEQ ID 304) GIRIAA 3′ Onc2F25′ GATGCAAGGTCGCATATGAGAATGGCATTTGTTG (SEQ ID 305) GGGTAA 3′ Onc2G25′ GATGCAAGGTCGCATATGAGAATGGCATTTGTTG (SEQ ID 306) GGGAAA 3′ Onc2H25′ GATGCAAGGTCGCATATGAGAATGGCATTTGTTG (SEQ ID 307) GCATAA 3′ Onc2I25′ GATGCAAGGTCGCATATGAGAATGGCATTTGTTG (SEQ ID 308) GGGCAA 3′ Onc2J25′ GATGCAAGGTCGCATATGAGAATGGCATTTGTTG (SEQ ID 309) GCACAA 3′ Onc2K15′ AATTCTAATACGACTCACTATAGGGAGAAGGTCA (SEQ ID 310) TATTCCTCMICATGIC 3′Onc2L1 5′ AATTCTAATACGACTCACTATAGGGAGAAGGTCA (SEQ ID 311)TATTCCTCAACATGIC 3′ Onc2M1 5′ AATTCTAATACGACTCACTATAGGGAGAAGGTCA (SEQ ID312) TATTCCTCIICATGTC 3′ Onc2N1 5′ AATTCTAATACGACTCACTATAGGGAGAAGGTCA(SEQ ID 313) TATTCCTCIICATGGC 3′ Onc2O15′ AATTCTAATACGACTCACTATAGGGAGAAGGTCA (SEQ ID 314) TATTCCTCIICATGAC 3′Onc2P1 5′ AATTCTAATACGACTCACTATAGGGAGAAGGTCA (SEQ ID 315)TATTCCTCIICATGCC 3′

TABLE 6 Preferred L1 PCR primers Primer name Sequence Onc2A1-PCR (SEQ ID149) 5′ AATGGCATTTGTTGGGGTAA 3′ Onc2A2-PCR (SEQ ID 150)5′ TCATATTCCTCCCCATGTC 3′ Onc2B1-PCR (SEQ ID 152)5′ AATGGCATTTGTTGGSRHAA 3′ Onc2B2-PCR (SEQ ID 153)5′ TCATATTCCTCMMCATGDC 3′ Onc2C1-PCR (SEQ ID 156)5′ AATGGCATTTGTTGGSIIAA 3′ Onc2D1-PCR (SEQ ID 157)5′ AATGGCATTTGTTGGIIHAA 3′ Onc2E1-PCR (SEQ ID 158)5′ AATGGCATTTGTTGGIRIAA 3′ Onc2F1-PCR (SEQ ID 159)5′ AATGGCATTTGTTGGGGTAA 3′ Onc2G1-PCR (SEQ ID 160)5′ AATGGCATTTGTTGGGGAAA 3′ Onc2H1-PCR (SEQ ID 161)5′ AATGGCATTTGTTGGCATAA 3′ Onc2I1-PCR (SEQ ID 162)5′ AATGGCATTTGTTGGGGCAA 3′ Onc2J1-PCR (SEQ ID 163)5′ AATGGCATTTGTTGGCACAA 3′ Onc2K2-PCR (SEQ ID 164)5′ TCATATTCCTCMICATGIC 3′ Onc2L2-PCR (SEQ ID 165) 5′ TCATATTCCTCAACATGIC3′ Onc2M2-PCR (SEQ ID 166) 5′ TCATATTCCTCIICATGTC 3′ Onc2N2-PCR (SEQ ID167) 5′ TCATATTCCTCIICATGGC 3′ Onc2O2-PCR (SEQ ID 168)5′ TCATATTCCTCIICATGAC 3′ Onc2P2-PCR (SEQ ID 169) 5′ TCATATTCCTCIICATGCC3′

The HPV-specific sequences in SEQ ID NOs:149 and 150 (primersOnc2A2/Onc2A1-PCR and Onc2A1/Onc2A2-PCR) are identical to fragments ofthe HPV type 16 genomic sequence from position 6596-6615 (SEQ ID NO:149;Onc2A2/Onc2A1-PCR), and from position 6729 to 6747(SEQ ID NO:150;Onc2A1/Onc2A2-PCR).

The HPV-specific sequences SEQ ID NOs:152 and 153 (Onc2B2/Onc2B1-PCR andOnc2B1/Onc2B2-PCR) are variants of the above sequences, respectively,including several degenerate bases. Representations of the sequences ofdegenerate oligonucleotide molecules provided herein use the standardIUB code for mixed base sites: N=G, A, T, C; V=G, A, C; B=G, T, C; H=A,T, C; D=G, A, T; K=G, T; S=G, C; W=A, T; M=A, C; Y=C, T; R=A, G.

It is also possible to use variants of the HPV-specific sequences SEQ IDNO:152 (Onc2B2/Onc2B1-PCR) and SEQ ID NO:153 (Onc2B1/Onc2B2-PCR) whereinany two of nucleotides “SRH” towards the 3′ end of the sequence arereplaced with inosine (I), as follows:

5′ AATGGCATTTGTTGGIIHAA 3′ (SEQ ID 157) 5′ AATGGCATTTGTTGGSIIAA 3′ (SEQID 156) 5′ AATGGCATTTGTTGGIRIAA 3′ (SEQ ID 158)

The HPV-specific sequences SEQ ID NOs: 156-163 (present in primersOnc2C2, Onc2D2, Onc2E2, Onc2F2, Onc2G2, Onc2H2, Onc212, Onc2J2,Onc2C1-PCR, Onc2D1-PCR, Onc2E1-PCR, Onc2F1-PCR, Onc2G1-PCR, Onc2H1-PCR,Onc211-PCR and Onc2J1-PCR) are variants based on the HPV-specificsequence SEQ ID NO:152 (Onc2B2/Onc2B1-PCR), whereas the HPV-specificsequences SEQ ID NOs: 164-169 (present in primers Onc2K1, Onc2L1,Onc2M1, Onc2N1, Onc201, Onc2P1, Onc2K2-PCR, Onc2L2-PCR, Onc2M2-PCR,Onc2N2-PCR, Onc202-PCR and Onc2P2-PCR are variants based on theHPV-specific sequence SEQ ID NO:153 (Onc2B1/Onc2B2-PCR). These variantsinclude degenerate bases and also inosine (I) residues. This sequencevariation enables oligonucleotides incorporating the variant sequencesto bind to multiple HPV types. Inosine bases do not interfere withhybridization and so may be included at sites of variation between HPVtypes in order to construct a “consensus” primer able to bind tomultiple HPV types.

Any one or more of primers Onc2A2, Onc2B2, Onc2C2, Onc2D2, Onc2E2,Onc2F2, Onc2G2, Onc2H2, Onc212 and Onc2J2, may be used in combinationwith any one or more of primers Onc2A1, Onc2B1, Onc2K1, Onc2L1, Onc2M1,Onc2N1, Onc201 and Onc2P1, for NASBA amplification of HPV L1 mRNA.

Any one or more of primers Onc2A1-PCR, Onc2B1-PCR, Onc2C1-PCR,Onc2D1-PCR, Onc2E1-PCR, Onc2F1-PCR, Onc2G1-PCR, Onc2H1-PCR, Onc2I1-PCRand Onc2J1-PCR, may be used in combination with any one or more ofprimers Onc2A2-PCR, Onc2B2-PCR, Onc2K2-PCR, Onc2L2-PCR, Onc2M2-PCR,Onc2N2-PCR, Onc202-PCR and Onc2P2-PCR for PCR amplification of HPV L1mRNA.

The invention will be further understood with reference to the followingexperimental examples and figures in which:

FIG. 1A shows the results of a single reaction real-time NASBA assayusing a FAM molecular beacon for HPV 16 on a patient sample while FIG.1B shows multiplexed real-time NASBA assay using the FAM molecularbeacon of FIG. 1A and a molecular beacon labeled with Texas red for UIA,

FIG. 2A shows single reaction real-time NASBA with FAM molecular beaconfor HPV 18 on a patient sample while FIG. 2B shows a multiplexed versionwith a Texas red labeled molecular beacon for HPV 18 and a FAM labeledbeacon for HPV 33,

FIG. 3A shows single reaction real-time NASBA with HPV 31 FAM labeledmolecular beacon while FIG. 3B shows multiplexed version including HPV45 Texas red labeled molecular beacon,

FIG. 4A shows single reaction real-time NASBA with HPV 33 FAM labeledmolecular beacon while FIG. 4B is a multiplexed version including aTexas red labeled HPV 18 molecular beacon,

FIG. 5A shows single reaction real-time NASBA with HPV45 FAM labeledmolecular beacon while FIG. 5B shows the multiplexed version includingHPV 45 Texas red labeled molecular beacon and a FAM labeled HPV 31molecular beacon, and

FIG. 6 shows HPV detected by PreTect HPV-Proofer and PCR compared tocytology or histology.

EXAMPLE 1 Detection of HPV mRNA by NASBA-Based Nucleic AcidAmplification and Real-Time Detection Collection and Preparation ofClinical Samples

Pap smears and HPV samples were collected from 5970 women in thecervical screening program in Oslo, Norway. Samples intended for RNA/DNAextraction were treated as follows:

Cervical samples were collected from each women attending the cervicalscreening program using a cytobrush (Rovers Medical Devices, TheNetherlands). The cytobrush was then immersed in 9 ml lysis buffer (5MGuanidine thiocyanate). Since RNA is best protected in the 5M guanidinethiocyanate at −70° C. only 1 ml of the total volume of sample was usedfor each extraction round. The samples in lysis buffer were stored at−20° C. for no more than one week, then at −70° C. until isolation ofDNA/RNA.

RNA and DNA were automatically isolated from 5300 women in the firstround of extraction, using 1 ml from the total sample of 9 ml in lysisbuffer. RNA and DNA were extracted according to the “Booms” isolationmethod from Organon Teknika (Organon Teknika B. V., Boselind 15, P.O.Box 84, 5280 AB Baxtel, The Netherlands; now Biomerieux, 69280 Marcyl'Etoile, France) using the Nuclisens™ extractor following the protocolfor automated extraction.

Cell Lines

DNA and RNA from HeLa (HPV 18), SiHa (HPV 16) and CaSki (HPV 16) celllines were used as positive controls for the PCR and NASBA reactions.These cells were also used as sample material in the sensitivity study(Example 2). SiHa cells have integrated 1-2 copies of HPV 16 per cell,whilst CaSki cells have between 60-600 copies of HPV 16, both integratedand in the episomal state. HeLa cells have approximately 10-50 copies ofHPV 18 per cell.

HPV Detection and Typing by PCR

Isolated DNA from cervical scrapes was subjected to PCR using theconsensus GP5+/6+ primers (EP-B-0 517 704). The PCR was carried out in50 μl reaction volume containing 75 mM Tris-HCl (pH 8.8 at 25° C.), 20mM (NH₄)₂SO₄, 0.01% Tween 20™, 200 mM each of dNTP, 1.5 mM MgCl₂, 1 Urecombinant Taq DNA Polymerase (MBI Fermentas), 3 μl DNA sample and 50pmol of each GP5+ and GP6+ primers. A 2 minutes denaturation step at 94°C. was followed by 40 cycles of amplification with a PCR processor(Primus 96, HPL block, MWG, Germany). Each cycle included a denaturationstep at 1 minutes, a primer annealing step at 40° C. for 2 minutes and achain elongation step at 72° C. for 1.5 minutes. The final elongationstep was prolonged by 4 minutes to ensure a complete extension of theamplified DNA.

The GP5+/6+ positive samples were subjected to HPV type 16, 31 and 33PCR protocols as follows:

HPV 16, 31 and 33: The PCR was carried out in 50 μl containing 75 mMTris-HCl (pH 8.8 at 25° C.), 200 mM each of dNTP, 1.5 mM MgCl₂, 2.5 Urecombinant Taq DNA Polymerase (MBI Fermentas), 3 μl DNA sample and 25pmol of each primers. A 2 minutes denaturation step at 94° C. wasfollowed by 35 cycles of amplification with a PCR processor (Primus 96,HPL block, MWG, Germany). Each cycle included a denaturation step at 30sec, a primer annealing step at 57° C. for 30 sec and a chain elongationstep at 72° C. for 1 minutes. The final elongation step was prolonged by10 minutes to ensure a complete extension of the amplified DNA. Theprotocol for HPV 33 had a primer annealing step at 52° C. HPV 18protocol:Primers were designed to identify HPV type 18. The PCR was carried outin 50 μl containing 75 mM Tris-HCl (pH 8.8 at 25° C.), 20 mM (NH₄)₂SO₄,0.01% Tween 20, 200 mM each of dNTP, 2.0 mM MgCl₂, 2.5 U recombinant TaqDNA Polymerase (MBI Fermentas), 3 μl DNA sample and 25 pmol of eachprimer. A 2 minutes denaturation step at 94° C. was followed by 35cycles of amplification in a PCR processor (Primus 96, HPL block, MWG,Germany). Each cycle included a denaturation step at 30 sec, a primerannealing step at 57° C. for 30 sec and a chain elongation step at 72°C. for 1 minutes. The final elongation step was prolonged by 10 minutesto ensure a complete extension of the amplified DNA.

A primer set directed against the human □-globin gene was used as acontrol of the DNA quality (Operating procedure, University HospitalVrije Universiteit, Amsterdam, The Netherlands). The PCR was carried outin 50 μl containing 75 mM Tris-HCl (pH 8.8 at 25° C.), 200 mM each ofdNTP, 1.5 mM MgCl₂, 1 U Recombinant Taq DNA Polymerase (MBI Fermentas),3 μl DNA sample and 25 pmol of each primer. A 2 minutes denaturationstep at 94° C. was followed by 35 cycles of amplification with a PCRprocessor (Primus 96, HPL block, MWG, Germany). Each cycle included adenaturation step at 94° C. for 1 minute, a primer annealing step at 55°C. for 1½ minutes and a chain elongation step at 72° C. for 2 minutes.The final elongation step was prolonged by 4 minutes to ensure acomplete extension of the amplified DNA. HeLa was used as positivecontrols for HPV 18, while SiHa or CaSki were used as positive controlfor HPV 16. Water was used as negative control.

Primers used for HPV PCR: Length Type Primer Primer sequence (SEQ IDNo.) Position (bp) HPV16 Pr1 5′ TCA AAA GCC ACT GTG TCC TGA 3′ (318)421-440 119 Pr2 5′ CGT GTT CTT GAT GAT CTG CAA 3′ (319) 521-540 HPV18Pr1 (5′ TTC CGG TTG ACC TTC TAT GT 3′) (320) 651-670 186 Pr2 (5′ GGT CGTCTG CTG AGC TTT CT 3′) (321) 817-836 HPV31 Pr1 5′ CTA CAG TAA GCA TTGTGC TAT GC 3′ (322) 3835-3875 153 Pr2 5′ ACG TAA TGG AGA GGT TGC AAT AACCC 3′ (323) 3963-3988 HPV33 Pr1 5′ AAC GCC ATG AGA GGA CAC AAG 3′ (324)567-587 211 Pr2 5′ ACA CAT AAA CGA ACT GTG TGT 3′ (346) 758-778 Gp+ Gp5+5′ TTT GTT ACT GTG GTA GAT ACT AC 3′ (338) 6624-6649 150 Gp6+ 5′ GAA AAATAA ACT GTA AAT CAT ATT C (339) 6719-6746 BGPCO3 Pr1 5′ ACA CAA CTG TGTTCA CTA GC (340) BGPCO5 Pr2 5′ GAA ACC CAA GAG TCT TCT CT (341)

Visualization of the PCR products was done on a DNA 500 chip (AgilentTechnologies, USA) according to their manual. The DNA chip uses microscale gel electrophoresis with an optimal detection limit of 0.5-50ng/ml. The results were interpreted using the Bioanalyzer 2100 software(Agilent Technologies, USA).

The following table confirms primers used for HPV PCR in patient samplesand indicates additional PCR primers useful for HPV 35, 39, 45, 51, 52,58 and HPV 6/11.

PCR primers for detection of HPV. Type Primer Primer sequence (SEQ IDNo.) Position Length (bp) HPV6/11 Pr1 5′ TAC ACT GCT GGA CAA CAT 3′(316) 514-531 123 Pr2 5′ TCA TCT TCT GAG CTG TCT 3′ (317) 619-636 HPV16Pr1 5′ TCA AAA GCC ACT GTG TCC TGA 3′ (318) 421-441 120 Pr2 5′ CGT GTTCTT GAT GAT CTG CAA 3′ (319) 520-540 HPV18 Pr1 5′ TTC CGG TTG ACC TTCTAT GT 3′ (320) 651-670 186 Pr2 5′ GGT CGT CTG CTG AGC TTT CT 3′ (321)817-836 HPV31 Pr1 5′ CTA CAG TAA GCA TTG TGC TAT GC 3′ (322) 3835-3857155 Pr2 5′ ACG TAA TGG AGA GGT TGC AAT AAC CC 3′ (323) 3964-3989 HPV33Pr1 5′ AAC GCC ATG AGA GGA CAC AAG 3′ (324) 567-587 212 Pr2 5′ ACA CATAAA CGA ACT GTG GTG 3′ (325) 758-778 HPV35 Pr1 5′ CCC GAG GCA ACT GACCTA TA 3′ (326) 610-629 231 Pr2 5′ GGG GCA CAC TAT TCC AA ATG 3′ (327)821-840 HPV39 Pr1 5′ GCA GAC GAC CAC TAC AGC AAA 3′ (328) 210-230 153Pr2 5′ ACA CCG AGT CCG AGT AAT A 3′ (329) 344-362 HPV45 Pr1 5′ GAA ACCATT GAA CCC AGC AGA AAA 3′ (330) 428-451 154 Pr2 5′ TTG CTA TAC TTG TGTTTC CCT ACG 3′ (331) 558-581 HPV51 Pr1 5′ GGA GGA GGA TGA AGT AGA TA 3′(332) 658-677 169 Pr2 5′ GCC CAT TAA CAT CTG CTG TA 3′ (333) 807-826HPV52 Pr1 5′ GTG CCT ACG CTT TTT ATC TA 3′ (334) 296-315 233 Pr2 5′ GGGGTC TCC AAC ACT CTG AAC A 3′ (335) 507-528 HPV58 Pr1 5′ TCA GGC GTT GGAGAC ATC 3′ (336) 157-174 162 Pr2 5′ AGC AAT CGT AAG CAC ACT 3′ (337)301-318 Gp+ Gp5+ 5′ TTT GTT ACT GTG GTA GAT ACT AC 3′ (338) 150 Gp6+5′ GAA AAA TAA ACT GTA AAT CAT ATT C (339) BGPCO3 Pr1 5′ ACA CAA CTG TGTTCA CTA GC (340) BGPCO5 Pr2 5′ GAA ACC CAA GAG TCT TCT CT (341)

NASBA RNA Amplification

Precautions for avoiding contamination:1. Perform nucleic acid release, isolation and amplification/detectionin separate laboratory areas.2. Store and prepare reagents for nucleic acid release, isolation andamplification/detection at the laboratory areas where nucleic acidrelease, isolation and amplification/detection are to be performed,respectively.3. Keep all tubes and vials closed when not in use.4. Pipettes and other equipment that have been used in one laboratoryarea must not be used in the other areas.5. Use a fresh pipette or pipette tip for each pipetting action.6. Use pipettes with aerosol resistant tips for fluids possiblycontaining nucleic acid. Pipetting of solutions must always be performedout of or into an isolated tube that is opened and closed exclusivelyfor this action. All other tubes and vials should be kept closed andseparated from the one handled.7. Use disposable gloves when working with clinical material possiblycontaining target-RNA or amplified material. If possible, change glovesafter each pipetting step in the test procedure, especially aftercontact with possibly contaminated material.8. Collect used disposable material in a container. Close and removecontainer after each test run.9. Soak tube racks used during nucleic acid isolation oramplification/detection in a detergent (e.g. Merck Extran MAO1 alkaline)for at least one hour after each test run.The following procedure was carried out using reagents from theNuclisens™ Basic Kit, supplied by Organon Teknika.Procedure for n=10 samples:—1. Prepare enzyme solution.Add 55 μl of enzyme diluent (from Nuclisens™ Basic Kit; containssorbitol in aqueous solution) to each of 3 lyophilized enzyme spheres(from Nuclisens™ Basic Kit; contains AMV-RT, RNase H, T7 RNA polymeraseand BSA). Leave this enzyme solution at least for 20 minutes at roomtemperature. Gather the enzyme solutions in one tube, mix well byflicking the tube with your finger, spin down briefly and use within 1hour. Final concentrations in the enzyme mix are 375 mM sorbitol, 2.5 μgBSA, 0.08 U RNase H, 32 U T7 RNA polymerase and 6.4 U AMV-reversetranscriptase.2. Prepare reagent sphere/KCl solution.For 10 samples: add 80 μl reagent sphere diluent (from Nuclisens™ BasicKit; contains Tris/HCl (pH 8.5), 45% DMSO) to the lyophilized reagentsphere (from Nuclisens™ Basic Kit; contains nucleotides, dithiotreitoland MgCl₂) and immediately vortex well. Do this with 3 reagent spheresand mix the solutions in one tube.Add 3 μl NASBA water (from Nuclisens™ Basic Kit) to the reconstitutedreagent sphere solution and mix well.Add 56 μl of KCl stock solution (from Nuclisens™ Basic Kit) and mixwell. Use of this KCl/water mixture will result in NASBA reactions witha final KCl concentration of 70 mM. Final concentrations in thereagent/KCl solution are 1 mM of each dNTP, 2 mM of ATP, UTP and CTP,1.5 mM GTP, and 0.5 mM ITP, 0.5 mM dithiotreitol, 70 mM KCl, 12 mMMgCl₂, 40 mM Tris-HCl (pH 8.5).3. Prepare primer/probe solution containing target-specific primers andmolecular beacon probe.For each target reaction transfer 91 μl of the reagent sphere/KClsolution (prepared in step 2) into a fresh tube. Add 25 μl ofprimers/molecular beacon probe solution (to give final concentration of˜0.1-0.5 μM each of the sense and antisense primers and ˜15-70 pmolmolecular beacon probe per reaction). Mix well by vortexing. Do notcentrifuge.In case less than 10 target RNA amplifications are being performed referto the table below for the appropriate amounts of reagent spheresolution, KCl/water solution and primers to be used. Primer solutionsshould be used within 30 minutes after preparation.

Reagent sphere Reactions (n) solution (μl) KCl/water (μl) Primer mix(μl) 10 80 30 10 9 72 27 9 8 64 24 8 7 56 21 7 6 48 18 6 5 40 15 5 4 3212 4 3 24 9 3 2 16 6 2 1 8 3 14. Addition of samplesFor each target RNA reaction:In a 96 well microtiter plate pipette 10 μl of the primer/probe solution(prepared in step 3) into each of 10 wells. Add 5 μl nucleic acidextract to each well. Incubate the microtiter plate for 4 minutes at65±1° C. Cool to at 41±0.5° C. for 4 minutes. Then to each well add 5 μlenzyme solution. Immediately place the microtiter plate in a fluorescentdetection instrument (e.g. NucliSens™ EasyQ Analyzer) and start theamplification.Results from Clinical Study

Table 7 shows the distribution of real-time NASBA HPV positive (L1and/or E6 expression) and PCR HPV positive cases related to cytologyresults. PCR amplification was carried out as described by Karlsen etal., J Clin Microbiol. 34: 2095-2100, 1996. The figures for expectedhistology are based on average results from similar study on CIN IIIlesions (Clavel et al., Br J Cancer, 84: 1616-1623, 2001). The resultsfrom several example cases are listed in Table 8.

TABLE 7 Normal Benign Condyloma CIN III Cytology 4474 66 16 15 PCR 9.0%44.6% 87.5% 73.3% Real-time   1% 24.6% 37.5% 73.3% NASBA Expected 0.2%5-15% 15-20%    71% Histology

TABLE 8 Internal No. Cytology PCR L1 NASBA E6 NASBA 84 Neg Neg Neg 31289 Neg 31 Pos 31 926 Neg Neg Pos 16 743 Benign Neg Neg 33 1512 Benign16 Pos 16 3437 Benign Neg Neg 18 3696 Benign 16 Pos Neg 2043 Condyloma16, 51 Pos 16 3873 Condyloma 16, 51 Pos 16 3634 CIN II 33 Neg 33 4276CIN III Neg Neg 18 4767 CIN III 18 Neg 18 1482 CIN III Neg Pos 16 5217CIN III 31 Neg 31 4696 CIN III Neg Neg Neg

EXAMPLE 2 Sensitivity of Real-Time NASBA on Control Cell Lines

Cervical cancer cell lines, CaSki, SiHa and HeLa were diluted in lysisbuffer either before automated extraction of nucleic acids using theBoom's extraction method from Organon Teknika/bioMerieux (parallels 1and 3), or after nucleic acid extraction (parallel 2). Real-time NASBAwas performed using molecular beacons probes labelled with Texas red(16, L1 and 18) or FAM (UIA, 33 and 31) following the protocol describedabove.

TABLE 9 CaSki CaSki HeLa Primer sets and probes 16 16 16 33 33 33 18 3118 31 18 31 E6 U1 E6 U1 E6 U1 L1 E6 L1 E6 L1 E6 E6 E6 E6 E6 E6 E6 Numberof Parallels Cells 1 1 2 2 3 3 1 1 2 2 3 3 1 1 2 2 3 3 100000 + + + + + + + − + − + − + − + − + −  10 000 + + + + + + + − + − +− + − + − + −  1 000 + + + + + + + − + − + − + − + − + −   100 + + + + + + + − + − + − + − + − + −    10 + + + + + + − − − − + − +− + − + −     1 − − + − + − − − − − − − + − + − + −    10⁻¹ − − − − − −− − − − − − − − − − − −Thus, it is possible to detect HPV E6 mRNA in less than 1 cell usingreal-time NASBA.Real-time NASBA was tested both as a multiplex assay and as singlereactions. The results from the following sensitivity study are based onparallel runs of CaSki, SiHa and HeLa cell lines, and on three parallelruns on synthetic DNA oligos for HPV type 16, 18, 31 and 33. Thedefinition of the detection limit is that both of the samples in theparallel are positive. The number in the brackets (x) indicates that thespecified amount of cells also have been detected in some runs.Sensitivity is defined as the amount of cells necessary for detection ofHPV in two parallel runs. The HPV types are determined from PCR and thespecificity is based on NASBA compared to PCR.

Sensitivity

PCR: the HPV consensus PCR using Gp5+/6+ detected only down to 104 SiHaand HeLa cells, and down to 103 CaSki cells. However, the type specificPCR primer-sets were more sensitive, detecting 103 (102) SiHa cells and0.1 CaSki cells for HPV 16 type specific PCR primer-set, while the HPV18 type specific PCR primer-set detected 102 HeLa cells.Real-time NASBA: Real-time NASBA with primers specific for UIA, detected10(1) SiHa and CaSki cells and 1 HeLa cell in the reaction mixture. Forthe HPV 16 specific primers, the lower detection limit was (10) (102, 1)SiHa cells and 10 (1) CaSki cells and for the HPV 18 specific primersthe detection limit was 1 (0.1) HeLa cell. The universal L1 primersdetected 10 CaSki cells. HeLa cells and SiHa cells were not detectedwith the universal L1 primers.Real-time multiplex NASBA with the UIA specific primers, had a lowerdetection limit of 10²(10) SiHa cells and 10(1) CaSki cells whencombined with the HPV 16 specific primers, which had a lower detectionlimit for 10(1) SiHa and 10(1) CaSki cells. The L1 specific primers incombination with the HPV 33 specific primers detected 10³(102) CaSkicells. There was no competing HPV 33 sample in the reaction. For the HPV18 specific primers, the lower detection limit was 1 (0.1) HeLa cellwhen combined with the HPV 31 specific primers. There was no competingHPV 31 sample in the reaction. Sensitivity of the HPV 31 and HPV 33specific primers were not tested, due to lack of cell lines harbouringthese HPV types. They were tested against samples containing HPV 31 andHPV 33, but the amount of cells and the copy number of HPV 31 and HPV 33in these cells were unknown and most probably varied in differentsamples.

TABLE 10 sensitivity of real-time NASBA compared to PCR NASBA PCR PrimerSiHa CaSki HeLa SiHa CaSki HeLa GP5+/6+ — — — 10⁴  10³ 10³ L1 — 10³(10²) — — — — U1A 10² (10)  10 (1) — — — — HPV 16  10 (1)  10 — 10³(10²)   0.1 — HPV 18 — — 1 (0, 1) — — 10²Real-time NASBA was performed on samples from women admitted to ØstfoldCentral Hospital for treatment of CIN in the period of 1999-2001 (seeexample 3). Molecular beacon probes labeled with FAM or Texas red wereused together with the nucleic acid extraction and NASBA protocolsdescribed above. The results are shown in FIGS. 1A and 1B (HPV16-patient sample 205), FIGS. 2A and 2B (HPV 18-patient sample 146),FIGS. 3A and 3B (HPV 31-patient sample 236), FIGS. 4A and 4B (HPV33-patient sample 218) and FIGS. 5A and 5B (HPV 45-patient sample 343).In each case, the “A” figure is a single reaction while the “B” figureis the multiplex assay.Specificity: Cross reactivity of Real-time NASBA. Real-time NASBA primercombinations were tested against 490 cervical samples from the Oslostudy positive with PCR for HPV 6/11, 16, 18, 31, 33, 35, 39, 45, 51,52, 58 or HPV X to check for cross reactivity between HPV types usingNASBA. All samples have been typed by consensus PCR and type specificPCR for the respective HPV types, except for HPV type 39(2), 52(1) and58(2). These samples are added to test against PreTect HPV-Proofer. HPVX are positive for consensus Gp5+/6+ PCR but negative for HPV6/11, 16,18, 31, 33, 35, 45 and 51 by type specific PCR. Results are shown intable 14. No cross-reactivity was shown. Sequence confirmation of aselected number of cases from table 14 is shown in table 14a.PCR: a total of 773 cervical samples were tested with PCR and thePreTect HPV-Proofer (Real time multiplex NASBA), and a total of 24.6%(190/773) samples were positive with the Gp5+/6+ consensus PCR primers.74.1% (83/112) were typed to be HPV 16, 13% (15/112) HPV 18, 17%(19/112) HPV 31 and 12% (13/112) HPV 33 including multiple HPVinfections. A total of 103 samples had single or multiple HPVinfections, and 91.3% (94/103) had only a single HPV infection. DoubleHPV infections occurred in 8.7% (9/103) of the samples. All samples werefirst tested with the consensus Gp5+/6+ PCR primers. The HPV PCRnegative samples from the consensus Gp5+/6+ were then tested withβ-globin control primers for a verification of intact DNA. The HPV PCRpositive samples were not subjected to this DNA control. The HPVnegative samples in this study were all positive with β-globin controlPCR primers. Only DNA samples positive with Gp5+/6+ PCR were subjectedto HPV type specific PCR. HPV types of interest were HPV 16, 18, 31 and33.Real-time multiplex NASBA: For the real-time NASBA reactions, theprimers and probes for the UIA gene product were used as a performancecontrol for intact RNA. Samples negative for UIA were rejected.A total of 14.2% (110/773) of the samples were positive with at leastone of the HPV type-specific NASBA primers including samples showingmultiple HPV infections. From these samples 54.5% (60/110) were positivewith HPV 16 NASBA primers, 13.6% (15/110) with HPV 18 primers, 21.8%(24/110) with HPV 31 primers and 13.6% (15/110) with HPV 33 primers. Atotal of 45 samples were positive with the L1 consensus primers andusually together with HPV 16 E6/E7 oncogene expression 82.2% (37/45).The consensus L1 was detected in 2.2% (1/45) together with either HPV18, 31 and 33 respectively. L1 was also detected alone in 8.9% (4/45)cases, and they all were PCR positive with Gp5+/6+ primers. A total of108 samples had single or multiple HPV infections, and 98.1% (106/108)had only a single HPV infections. Double mRNA expression occurred in1.9% (2/108) of the samples.Real-time multiplex NASBA compared to PCR: a total of 87 samples showedpresence of HPV 16 DNA or RNA with HPV 16 PCR or PreTect HPV-Proofer.64.4% (56/87) were determined to be positive for HPV 16 with both PCRand real-time NASBA. 39.1% (34/87) were only positive with PCR and 3.4%(3/87) were positive only with real-time NASBA. For HPV 18, a total of20 samples showed presence of HPV 18 DNA or RNA with either PCR orreal-time NASBA. From these 20 samples, 50% (10/20) were positive withboth tests, and 35% (7/20) were only positive with PCR and 15% (3/20)were only positive with real-time NASBA. A total of 27 samples showedpresence of HPV 31 DNA or RNA with either PCR or real-time NASBA. Out ofthese 27 samples, 59.3% (16/27) were positive with both tests and 11.1%(3/27) were positive only with the PCR test and 18.5% (5/27) were onlypositive with the real-time NASBA test. For HPV 33, a total of 18samples showed presence of HPV DNA or RNA with either PCR or PreTect-HPVProofer and 55.6% (10/18) of the samples were tested positive with bothtests. 16.7% (3/18) were only positive with PCR and 22.2% (4/18) wereonly positive with real-time NASBA.

TABLE 11 statistical distribution of HPV in samples with PCR andreal-time NASBA PCR % NASBA % Total samples 773 773 Total positive 19024.6 110 14.2 samples HPV16 83 74.1 60 54.5 HPV 18 15 13 15 13.6 HPV 3119 17 24 21.8 HPV33 13 12 15 13.6 HPV X 78 69.6 — —

TABLE 12 correspondence between PCR and real-time NASBA Both Only % %Only % Total tests % PCR + (PCR) (Total) NASBA % NASBA (total) HPV 16 8756 64.4 34 41.0 39.1 3 5 3.4 HPV 18 20 10 50.0 7 46.7 35.0 3 20 15.0 HPV31 27 16 59.3 3 15.8 11.1 5 20.8 18.5 HPV 33 18 10 55.6 3 23.1 16.7 426.7 22.2

TABLE 13 Real-time NASBA results for L1 Total % L1 (NASBA) 45 100 L1 +HPV 16 37 82.2 L1 alone 4 8.9 L1 + HPV 18 1 2.2 L1 + HPV 31 1 2.2 L1 +HPV 33 1 2.2

TABLE 14 Genetic specificity of real-time multiplex NASBA compared toPCR NASBA HPV (PCR) Primers 6/11 16 18 31 33 35 39 45 51 52 58 X TotalNumber 16 2 28 1 0 1 1 0 0 5 0 0 2 18 1 1 18 0 1 0 0 0 1 0 0 1 31 1 0 113 1 5 0 1 1 0 0 0 33 1 2 0 2 12 2 0 1 2 0 0 1 45 2 1 1 1 1 1 0 17 1 0 01 Sum 43 71 36 32 25 23 2 23 31 1 2 201 490 tested

TABLE 14a DNA sequencing from Gp5+ PCR primers (not to be included inthe article) HPV type by PreTect HPV- HPV type by Int No HPV type by PCRProofer Sequencing (BLAST) 1272 16 16 16 152 35 35 2655 58 58 2924 33 3333 2942 18 18 18 2987 16 16 16 3016 33 33 33 3041 35 35 3393 35 35 387316 16 16 4767 18 18 18 5707 18 18 18 845 X 39

Discussion

Sensitivity of real-time NASBA was generally better than the sensitivityof PCR. The general sensitivity of real-time NASBA for all the markerswere between 1 and 10² cells, which is considerable better than for thePCR reaction with a sensitivity range from 102 to 10⁴. As expected, thesensitivity of the specific primers and probes were better than thesensitivity of the universal primers and probes. Real-time NASBA wasjust as sensitive or more sensitive than real-time multiplex NASBA.

Real-time NASBA primers and molecular beacon probe directed towards UIA(a human house keeping gene) were used as a performance control of thesample material in the real-time NASBA reaction to ensure that the RNAin the sample material was intact. A positive signal from this reactionwas necessary for a validation of the real-time NASBA reaction.

The sensitivity of the universal real-time NASBA with L1 (the majorcapsid protein of HPV) was much better than for the universal Gp5+/6+PCR, also directed against L1, with a sensitivity of 10 cells comparedto 10³ (102) CaSki cells. These two primer sets (PCR and NASBA) havetheir targets in the same region of the conserved L1 gene of differentHPV types. The differences in sensitivity may be due to the fact thatthere is usually one copy of each gene per cell, while the copy numberof mRNA may be several hundreds. The real-time NASBA L1 primers did notdetect SiHa or HeLa cells as the Gp5+/6+ PCR primers did, indicatinglack of L1 expression in these cell lines. Gp5+/6+ PCR primers detected10⁴ SiHa or HeLa cells. Considering the amount of HPV copies in eachcell, it makes sense that the CaSki cells were detected in 1/10 theamount of cells from SiHa and HeLa since CaSki cells have 60-600 HPVcopies per cell, both integrated and episomal, while SiHa cells have 1-2HPV copies integrated per cell and HeLa cells have 10-50 HPV copiesintegrated per cell. The L1 primer set detected only CaSki cells, withboth integrated and episomal forms of HPV, and not in SiHa or HeLacells, with only integrated forms of HPV. This might indicate that theL1 gene is only expressed in episomal states of HPV infection, andtherefore L1 may be a valuable marker for integration and persistence ofHPV infection.

The HPV type-specific NASBA primers are directed against the full lengthE6/E7 transcript, which are expressed in large amount in cancer cellsdue to lack of E2 gene product. The real-time NASBA 16 type specificprimers detected 10(1) SiHa cells and 10(1) CaSki cells compared to HPV16 PCR primers that detected 10³(10²) SiHa cells. The explanation forthis might be the different amount of HPV copies in each cell line. TheCaSki cells have both integrated and episomal forms of HPV, while SiHahas only integrated forms of HPV. This may be due to high expression ofmRNA from the E6/E7 genes. For detection of CaSki cells, the detectionlimit for the NASBA HPV 16 primers were 10(1) CaSki cells compared to0.1 CaSki cells for the HPV 16 PCR primers. This is peculiar, but anexplanation may be that the CaSki cells contain from 60-600 copies ofHPV 16 DNA, so that it is possible to detect 0.1 CaSki cells with 6-60HPV 16 DNA copies. The lower sensitivity of real-time NASBA compared toPCR may indicate that the expression of E6/E7 in the CaSki cells ismoderate/low. Degradation of the unstable mRNA may also be anexplanation. The amount of HPV copies in the CaSki cells may be in theorder of 60-600 times more than in the SiHa cells, which is shown by themore sensitive detection of CaSki cells.

The type specific HPV 18 PCR primers detected 102 HeLa cells. This is amagnitude of 100 better than the HPV consensus Gp5+/6+ primers andstates that specific primers are generally more sensitive than consensusprimers. The sensitivity of the type specific HPV 18 NASBA primers was 1(0.1) HeLa cells, indicating high expression of E6/E7 in HeLa cells.

The sensitivity of UIA NASBA primers was 10 SiHa or CaSki. The targetfor the UIA primer set is a human housekeeping gene that is expressed inevery human cell.

The sensitivity of PCR and NASBA varies for different primer sets andsample material, and generally type specific primers are more sensitivethan consensus primers due to base pair mismatch in consensus primersets. The annealing temperature for the primers in the PCR reaction canbe optimised, giving optimal reaction condition for the primers. Incontrast to the annealing temperature in PCR, the annealing temperaturefor the NASBA primers must be fixed at 41° C. This lack of temperatureflexibility may make the NASBA primers less sensitive and specific thanthe PCR primers.

PCR amplifies double stranded DNA and the target is usually present asone copy per cell and this makes it vulnerable to the number of cells inthe sample material. The target for the NASBA reaction is RNA, and mRNAmay be present as multiple copies per cell, depending on the expressionof the genes. By choosing a gene that is highly expressed, the mRNA copynumber may be several hundred per cell and therefore easier to detect.

dsDNA is relatively stable in the cell and the material stays intact fora long time. In contrast to dsDNA, mRNA is generally not very stable anddegradation of mRNA is rapid depending on the cell. There is no detectedDNase or RNase activity in the lysis buffer so both dsDNA and ssRNAshould be stable. Autocatalytic activity may degrade both DNA and RNA.The DNA/RNA from the cervix sample should stay intact, when stored inthe lysis buffer, for 24 hours at 15-30° C., 7 days at 2-8° C. or at−70° C. for long term storage.

A limitation in the real-time NASBA reaction is the concentration of themolecular beacon probes. The amount of products will exceed theconcentration of the molecular beacon probes and therefore it will notbe detected because a high molecular beacon probe concentration willmake the reaction mixture more complex and inhibit the amplificationreaction.

Nucleotides may also be a limitation to the final amount of theamplification product, both in the PCR and in the NASBA reaction. Thefinal concentration of the amplified product may in itself inhibitfurther amplification because of the amount of product and thecomplexity of the reaction mixture. During a NASBA reaction in thepresence of molecular beacons, the probe might compete with theamplification by hybridising to the template, making it unavailable forfollowing RNA synthesis. In this way, RNA is subtracted as substrate forthe reverse transcription steps and further RNA synthesis by T7 RNApolymerase. This competition is not significant with low amounts ofmolecular beacon, and with a high amount of molecular beacon thisinhibition can be overcome by a higher number of copies of input RNA.

The linear relationship between the amount of input RNA and thetime-to-positive signal was tested in a ten-fold serial dilutions ofdifferent HPV cell lines. There was a clear indication that a positivesignal was dependent on the amount of input RNA and time. The multiplexreaction needed more time than the single reaction to show a positivesignal. This might be due to competition in the more complex mixture inthe multiplex reaction vessel and also to the fact that the multiplexreaction has a different and lower concentration of primer and probe.The relationship between amount of target RNA and time to positivesignal opens up for a real-time multiplex quantitative amplificationreaction with internal RNA standards in each reaction vessel.

Real-time NASBA: single vs. multiplex. Real-time NASBA was generallymore sensitive than real-time multiplex NASBA. This was as expectedbecause of competition between primers and probes in the multiplexreaction. The final concentration of primers and molecular beacon probeswere optimised in the multiplex reaction so that for at least one of theprimer and probe sets the concentration were lower than in the singlereaction. From this it follows that with a lower concentration ofprimers, the less sensitive the reaction, or at least the less rapid thereaction. It will take longer time to reach the exponential stage of theamplification reaction and therefore longer time to detect the products.The concentration of the primers will not be a limitation to the finalconcentration of the product in the NASBA reaction because the doublestranded DNA created from the primers will continue to serve as atemplate for the RNA polymerase over and over again in a loop. Thesensitivity of multiplex real-time NASBA was the same for HPV 16 and HPV18 compared to single real-time NASBA, but the sensitivity for L1decreased drastically from a detection limit of 10(1) in the singlereaction to 10³(10²) CaSki cells in the multiplex reaction. For UIANASBA primers, the sensitivity decreased from 10(1) to 10²(10) SiHacells, while the detection limit remained the same for the CaSki cells.This decrease in detection limit may be to more complex competition ofprimers and molecular beacon probes in the multiplex reaction. The finalconcentration of primers and molecular beacon probes may not be the bestand the different primers and molecular beacon probes in the multiplexreaction may interfere with each other. The UIA NASBA primers detected 1HeLa cell. One might expect the same detection level in all the celllines, but the sensitivity of HeLa cells were 1/10 of the detectionlevel of SiHa and CaSki cells. These cell lines are cancer cells andthey might have different impact on the cells so that the expression ofUIA is different. The differences may also be due to different amount ofcells in each reaction, because of counting errors during harvesting ofthe cells.

Real-time NASBA showed no cross reactivity between HPV 16, 18, 31 and 33or with HPV 6/11, 35, 39, 45, 51, 52, 58 or HPV X.

The specificity of the PCR reaction may be better than the specificityof the real-time NASBA reaction because the NASBA reaction is anisothermal reaction at 41° C. with no possibilities to change theannealing temperature of the primers. The primers are basically designedthe same way as for the PCR primers. In a PCR reaction, you have thepossibility to change the annealing temperature, in contrast to theNASBA reaction, and therefore choose an annealing temperature that isoptimal for the two primers. This makes the annealing of the primersmore specific. The PCR results where visualized with gelelectrophoresis. But the molecular beacon probes in the real-time NASBAreaction is an additional parameter compared to PCR and therefore maygive the overall NASBA reaction a better specificity. It is also easierto find two different regions on the DNA sequence for primer annealingbecause there is much greater flexibility in the length of the PCRproduct, than for the NASBA product, which should be less than 250 bp.It is important for the specificity of the NASBA reaction to choose aunique area that is not conserved among the different HPV types. Acouple of base pair mismatches may still give an amplification orhybridisation of the target.

Detection of CaSki (integrated and episomal state) cells with theuniversal L1 NASBA primers and not SiHa or HeLa (both integrated) maygive an indication that integrated HPV doesn't show any L1 expression,while HPV in the episomal state may have L1 expression.

In summary, an identification assay has been developed for HPV type 16,18, 31 and 33 that can accurately identify the oncogenic E6/E7expression of these HPV types. The assay can also identify theexpression of the major capsid protein, L1.

EXAMPLE 3 Further Clinical Study in 190 Patients Patients/ClinicalSamples

Biopsies from 190 women admitted to Østfold central-hospital fortreatment of CIN in the period 1999-2001. The mean age of the 190 womenincluded in the study was 37.4 years (range 22-74 years). Biopsies werefrozen in −80° C. immediately after collection.

Cytological Examination of Samples

The routine cytological reports were used to record cytologicalfindings. No attempt was made to re-evaluate the slides. Each one ofthem indicated a CIN II-III condition, i.e. a high grade dysplasia orHSIL, which was the basis for hospital admittance, colposcopy andbiopsy.

Histological Examination of Samples

A biopsy, here termed biopsy 1, was taken after a high-grade cytologyreport. If it confirmed a high-grade lesion (CIN II or III), the patientwas again admitted to hospital, this time for colposcopically guidedconization. Before the conization, but after local anesthesia wasapplied, a second biopsy (biopsy 2) was taken from an area of portiowhere a dysplasia was most likely to be localised, judged from the grossfindings. This biopsy (2×2 mm) was frozen within 2 minutes in a −80° C.freezer.

Biopsy 2 was split in two when frozen and half was used for DNA/RNAextraction. The other half was fixed in 10% buffered formaldehyde andprocessed for histopathological examination. Some lesions were notcorrectly oriented in the paraffin block and had to be reoriented orserial sectioned in order to show the relevant surface epithelium.Consequently, it cannot be guaranteed that exactly the same tissue wasused for the extraction and for the histopathological evaluation. Thecone specimen, finally, was evaluated by the local pathologist, who inall cases could confirm the presence of dysplasia. It was not always thesame grade as in the original biopsy, and, in many cases, not the sameas in biopsy 2.

Extraction of Nucleic Acids

Nucleic acids were isolated using the automated Nuclisens Extractor aspreviously described (Boom et al., 1990). Each biopsy was cut in twopieces, one intended for histological examination and the other half forRNA analysis. The material intended for RNA analysis was divided intosmaller pieces while kept on dry ice (−80° C.) and put into 1 ml oflysisbuffer (as above) followed by 20 seconds of homogenisation usingdisposable pestles. 100 ml of the sample was further diluted 10 fold inlysisbuffer and 100 ml was then extracted for DNA/RNA. The extractedDNA/RNA was eluted with ˜40 ml of elution buffer (Organon Teknika) andstored at −70° C.

All molecular beacon probes used in this study employ the fluorophoreFAM (6-carboxyfluorescein) at the 5′ end of the structure. This wasbound to a variable stem-loop sequence coupled to the universal quencher4-(4′dimethylaminophenylazo)benzoic acid (DABCYL) at the 3′ end. Theprobes were delivered by Eurogentec, Belgium. Final concentration of MBsused in the reaction was 2.5 mM. For the real-time NASBA we made use ofthe NucliSens Basic Kit (Organon Teknika, Netherlands), intended for thedevelopment of user-defined RNA amplification assays. The NASBAamplification was carried out in a volume of 20 μl. The primer-sets andprobes were directed against full-length E6/E7 mRNA for the high-riskHPV 16, 18, 31, and 33. As performance control, to avoid false negativeresults due to degradation of nucleic acid, we used a primer set andprobe directed against the human U1 small nuclear ribonucleoprotein(snRNP) specific A protein (UIA mRNA) (Nelissen et al., 1991). Allsamples were run in duplicate on separate machines (microplate readersfor measuring fluorescence and absorbance, Bio-tek FL-600 FA from MWG).mRNA isolated from CaSki/SiHa or HeLa cells served as positive controlsfor HPV 16 and HPV 18 transcripts, respectively. Negative controls,included for every 7 reaction, consisted of a reaction containing allreagents except mRNA.

HPV DNA Analysis; Polymerase Chain Reaction

The same extracts and amounts as used in the NASBA reaction were usedfor PCR. The L1 consensus primers Gp5+/Gp6+ were used to detect allsamples containing HPV DNA. The PCR amplification was carried out asdescribed above. The first DNA denaturation was done for 2 minutes at94° C., then 40 cycles of PCR were run: denaturation 1 minute at 94° C.,annealing for 2 minutes at 40° C., extension for 1.5 minutes at 72° C.,followed by a final extension for 4 minutes at 72° C. Typing of HPV wasperformed by using PCR type-specific primers against HPV 16, 18, 31, and33 (6/11, 35, 45, 51, 52, 58), as described above.

Results

Originally 190 patients were biopsied after being given the diagnosisCIN I, CINII, or CIN III by cytology. A high-grade lesion was confirmedby histologically examination, 150 samples diagnosed as CIN III (78.9%).Biopsy 2, taken before conization, was used for RNA analysis. However,histological examination of this biopsy diagnosed only 53 samples of theoriginally 150 as CIN III [54 were given no diagnosis, 24 diagnosed asCIN II, 18 as CIN I, and 4 as HPV/condylom]. The number of CIN IIsamples increased from 16 (8.4%) to 30 (15.8%) [by Histology 124diagnosed as CIN III, 4 as CIN II, 1 as carcinom, and 1 as CIN I. 12 CINII cases from Histology I were given a lower diagnosis in Histology II].The degree of CIN I increased from 6 samples (3.2%) to 32 samples(16.8%). The 2 squamous cell carcinomas were in Histology II diagnosedas CIN III, the adenocarcinom as CIN II. In 71 samples (38.4%)high-grade lesions were not detected.

HPV oncogenic RNA was detected in 69 (36%) of the 190 patients. Of the53 samples (28%) diagnosed as CIN III in Histology II, we found 40 (76%)cases showing HPV 16, 18, 31, or 33 oncogenic expression. In addition,we found oncogenic expression in 9 of 30 cases (30%) of CIN II, in 4 of32 cases (13%) of CIN I, in 14 of 71 cases (20%) not showing cellabnormalities, and in 2 of 4 (50%) samples diagnosed as HPV/condyloma.

HPV 16 RNA was found in 42 of the 190 patients, HPV 18 was found in 7(3.7%), HPV 31 in 15 (7.9%), and HPV 33 in 8 (4.2%). One patient hadmixed infection with HPV 16 and HPV 18, and one with HPV 16 and HPV 31.

Using the consensus Gp5+/Gp6+ primers directed against the L1 gene,encoding the major capsid protein, PCR detected HPV in 81 of the 190cervical biopsies (43%). Of the 119 cases given a diagnosis in thesecond histological examination (115 diagnosed as CIN, 4 asHPV/condyloma) 63 were found to contain HPV DNA. The additional 18 casesdetected were not given any histological diagnosis. 20 of the 81 caseswere not detected by NASBA; 7 out of these were given the diagnosis CINIII, 2 were diagnosed as CIN II, 4 diagnosed as CIN I, and 7 given nodiagnosis.

Type-specific PCR detected 85 cases containing HPV; 66 having HPV 16, 10HPV 18, 14 HPV 31, 7 HPV 33. 12 cases had multiple infection: 3 with HPV16+18; 4 with HPV 16+33, 5 with HPV 16+31. 20 no diagnosis.

EXAMPLE 4 HPV Detected by PreTect HPV-Proofer and PCR Compared toCytology and Histology

Normal and ASCUS samples (including borderline smears) were determinedby cytology. All samples were tested with consensus PCR and PreTectHPV-Proofer but only the consensus positive samples were typed by PCR.The CIN 3 and cancer samples were determined by histology and all thesamples were tested with all three methods. The results are shown inFIG. 6. Concordance between real-time multiplex NASBA and PCR comparedto cytology or histology is shown in Table 15 below.

TABLE 15 Concordance between real-time multiplex NASBA and PCR comparedto cytology or histology Cytology/Histology Concordance^(a) (Number)Concordance^(b) (Number) Normal 98.2% (4043) 42.8% (138) ASCUS^(c) 94.5%(55) 78.6% (14) CIN 3 94.3% (53) 93.2% (44) Cancer 99.0% (196) 98.8%(170) Only samples positive by Gp5+/6+ PCR have been typed.^(a)Including PCR and real-time multiplex NASBA positive and negativesamples. ^(b)Including only PCR and/or real-time multiplex NASBApositive samples. ^(c)ASCUS excluding borderline smears.

EXAMPLE 5

The invention provides a kit for detection of mRNA transcripts from theE6 gene(s) of HPV the kit comprising one or more of, two or more of andpreferably all of the following primer pairs and accompanyingidentification probes.

HPV16:HPV16.txt 7905 b.p HPV16P2: p2:116 (20) (SEQ ID No. 175)GATGCAAGGTCGCATATGAGCCACAGGAGCGACCCAGAAA 16 p1 (no7) (SEQ ID No. 177)AATTCTAATACGACTCACTATAGGGAGAAGG ATT CCC ATC TCT ATA TAC TA (51 baser)HPV16P02: p0:230 (20) (SEQ ID No. 19) TATGACTTTGCTTTTCGGGA H16e6702poHPV 18:HPV18.txt 7857 b.p HPV18P2: p2:698 (22) (SEQ ID No. 196)GATGCAAGGTCGCATATGAGGAAAACGATGAAATAGATGGAG H18e6702p2 HPV18P4: p1:817(20) (SEQ ID No. 203) AATTCTAATACGACTCACTATAGGGAGAAGGGGTCGTCTGCTGAGCTTTCT H18e6703p1 (Multiplex) HPV18P02: po:752 (21) (SEQ ID No. 32)GAACCACAACGTCACACAATG H18e6702po HPV 31:HPV31.txt 7912 b.p HPV31P3:p2:617 (20) (SEQ ID No. 210) GATGCAAGGTCGCATATGAGACTGACCTCCACTGTTATGAH31e6703p2 p1:766 (20) (SEQ ID No. 211)AATTCTAATACGACTCACTATAGGGAGAAGGTATCTACTTGTGTGCTCTG T H31e6703p1HPV31P04: po:686 (26) (SEQ ID No. 50) GGACAAGCAGAACCGGACACATCCAAH31e6704po HPV33:HPV33.txt 7909 b.p HPV33P1: p2:618 (22) (SEQ ID No.221) GATGCAAGGTCGCATATGAGTATCCTGAACCAACTGACCTAT H33e6701p2 p1:763 (19)(SEQ ID No. 222) AATTCTAATACGACTCACTATAGGGAGAAGGTTGACACATAAACGAACTGH33e6701p1 HPV33PO3: po:699 (23) (SEQ ID No. 59) GGACAAGCACAACCAGCCACAGCH33e6703poAs alternative to the probes shown above the kit may optionally includeone or more of the following molecular beacon probes:

Molecular Beacon Probes: H16e6702mb2-FAM (SEQ ID No. 175)ccagctTATGACTTTGCTTTTCGGGAagctgg H18e6702mb1-TxR (SEQ ID No. 198)cgcatgGAACCACAACGTCACACAATGcatgcg H31e6704mb2-FAM ((SEQ ID No. 215)ccgtcgGGACAAGCAGAACCGGACACATCCAAcgacgg H33e6703mb1-FAM (SEQ ID No. 225)ccaagcGGACAAGCACAACCAGCCACAGCgcttggPreferably the kit of the invention also includes the following primerpair and probe.:

HPV45: HPV45.txt 7858 bp (X74479)

HPV45P1: p2:430 (21): (SEQ ID No. 261)GATGCAAGGTCGCATATGAGAACCATTGAACCCAGCAGAAA H45e6701p2 p1:527(22): (SEQ IDNo. 262) AATTCTAATACGACTCACTATAGGGAGAAGGTCTTTCTTGCCGTGCCTGG TCAH45e6701p1 HPV45P01: po:500 (20): (SEQ ID No. 108) GTACCGAGGGCAGTGTAATAH45e6701poThe HPV 45 probe above may be replaced by an HPV molecular beacon probeas follows:

H45e6701mb1 cgatcgGTACCGAGGGCAGTGTAATAcgatcg (SEQ ID No. 175)In addition the kit may include one or more of the following primerpairs and accompanying identification probes depending on thegeographical area of use of the kit.

HPV52: HPV52.txt 7942 bp (X74481) HPV52P1: p2:144 (22): (SEQ ID No. 239)GATGCAAGGTCGCATATGAGTTGTGTGAGGTGCTGGAAGAAT H52e6701p2 p1:358(18): (SEQID No. 240) AATTCTAATACGACTCACTATAGGGAGAAGGCCCTCTCTTCTAATGTTT H52e6701p1HPV52P01: Po:296 (20): (SEQ ID No. 334) GTGCCTACGCTTTTTATCTA H52e6701poHPV58 HPV58.txt 7824 bp (D90400) HPV58P2: p2:173 (18): (SEQ ID No. 245)GATGCAAGGTCGCATATGAGTCTGTGCATGAAATCGAA H58e6702p2 p1:291 (18): (SEQ IDNo. 346) AATTCTAATACGACTCACTATAGGGAGAAGGAGCACACTTTACATACTG H58e6702p1HPV58P02: po:218 (22): (SEQ ID No. 84) TTGCAGCGATCTGAGGTATATG H58e6702poHPV51 HPV51.txt 7808 bp (M62877) HPV51PA/P: p2:655 (23): (SEQ ID No.275) GATGCAAGGTCGCATATGAG AGA GGA GGA GGA TGA AGT AGA TA H51e6702p2p1:807 (20): (SEQ ID No. 274) AATTCTAATACGACTCACTATAGGGAGAAGG GCC CATTAA CAT CTG CTG TA H51e6701p1 HPV51POA: po:771 (24): (SEQ ID No. 129)TGG CAG TGG AAA GCA GTG GAG ACA H51e67o2poThe probes shown above may be replaced in the kit by the followingmolecular beacon probes:

H52e6701mb1 (SEQ ID No. 343) cgatcgGTGCCTACGCTTTTTATCTAcgatcgH58e6702mb1 (SEQ ID No. 344) ccgtcgTTGCAGCGATCTGAGGTATATGcgacggH51e6702mb1 (SEQ ID No. 345) cgatcgTGG CAG TGG AAA GCA GTG GAG ACAcgatcg

1. An in vitro method of screening a human subject for the presence ofintegrated HPV or a modified episomal HPV genome, which method comprisesscreening the human subject for expression of full length mRNAtranscripts from the E6 gene of human papillomavirus, wherein a subjectpositive for expression of full length E6 mRNA from at least one HPVtype are scored as carrying integrated HPV or a modified episomal HPVgenome.
 2. The method of claim 1 wherein the human subject is previouslyidentified as infected with human papillomavirus DNA in cells of thecervix.
 3. The method of claim 1 wherein the human subject has aprevious diagnosis of ASCUS, CIN 1 lesions or condyloma.
 4. The methodof claim 1 which comprises screening for expression of full length E6mRNA transcripts from at least HPV type 16, wherein a subject positivefor expression of full length E6 mRNA from at least HPV type 16 isscored as carrying integrated HPV or a modified episomal HPV genome. 5.The method of claim 4 wherein said full length E6 mRNA transcripts ofHPV type 16 contain all of the region from nucleotide 97 to nucleotide880 in the E6 open reading frame, inclusive of nucleotides 97 and 880.6. The method of claim 1 wherein screening for E6 mRNA expression iscarried out using an amplification reaction to amplify a region of themRNA, together with real-time detection of the products of theamplification reaction.
 7. The method of claim 6 wherein screening forE6 mRNA expression is carried out using real-time NASBA.
 8. An in vitromethod of identifying human subjects having abnormal cell changes in thecervix, which method comprises screening a human subject for expressionof full length mRNA transcripts of the E6 gene of HPV, wherein a humansubject positive for expression of full length E6 mRNA from at least oneHPV type is identified as having abnormal cell changes in the cervix. 9.The method of claim 8 wherein the human subject is previously identifiedas infected with human papillomavirus DNA in cells of the cervix. 10.The method of claim 8 wherein the human subject has a previous diagnosisof ASCUS, CIN 1 lesions or condyloma.
 11. The method of claim 8 whichcomprises screening for expression of full length E6 mRNA transcriptsfrom at least HPV type 16, wherein a subject positive for expression offull length E6 mRNA from at least HPV type 16 is scored as carryingintegrated HPV or a modified episomal HPV genome.
 12. The method ofclaim 11 wherein said full length E6 mRNA transcripts of HPV type 16contain all of the region from nucleotide 97 to nucleotide 880 in the E6open reading frame, inclusive of nucleotides 97 and
 880. 13. The methodof claim 8 wherein screening for E6 mRNA expression is carried out usingan amplification reaction to amplify a region of the mRNA, together withreal-time detection of the products of the amplification reaction. 14.The method of claim 13 wherein screening for E6 mRNA expression iscarried out using real-time NASBA.